Methods and compositions for enhancing the efficacy and specificity of rna silencing

ABSTRACT

The present invention provides methods of enhancing the efficacy and specificity of RNA silencing. The invention also provides compositions for mediating RNA silencing. In particular, the invention provides siRNAs, siRNA-like molecules, shRNAs, vectors and transgenes having improved specificity and efficacy in mediating silencing of a target gene. Therapeutic methods are also featured.

RELATED APPLICATIONS

This application is a continuation-in-part of co-pending U.S. UtilityApplication Ser. No. 10/859,321, entitled “Methods and Compositions forEnhancing the Efficacy and Specificity of RNAi” (filed Jun. 2, 2004),which claims the benefit of U.S. Provisional Patent Application Ser. No.60/475,331, entitled “Methods and Compositions for Enhancing theEfficacy and Specificity of RNAi,” filed Jun. 2, 2003; U.S. ProvisionalPatent Application Ser. No. 60/507,928 entitled “Methods andCompositions for Enhancing the Efficacy and Specificity of RNAi,” filedSep. 30, 2003; and U.S. Provisional Patent Application Ser. No.60/575,268 entitled “Methods and Compositions for Enhancing the Efficacyand Specificity of RNAi,” filed May 28, 2004. The entire contents of theabove-referenced patent applications are incorporated herein by thisreference.

GOVERNMENT RIGHTS

This invention was made with government support under grant nos.GM062862, GM065236 and NS044952 awarded by the National Institutes ofHealth. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

Two types of ˜21 nt RNAs trigger post-transcriptional gene silencing inanimals: small interfering RNAs (siRNAs) and microRNAs (miRNAs). BothsiRNAs and miRNAs are produced by the cleavage of double-stranded RNA(dsRNA) precursors by Dicer, a nuclease of the RNase III family ofdsRNA-specific endonucleases (Bernstein et al., 2001; Billy et al.,2001; Grishok et al., 2001; Hutvàgner et al., 2001; Ketting et al.,2001; Knight and Bass, 2001; Paddison et al., 2002; Park et al., 2002;Provost et al., 2002; Reinhart et al., 2002; Zhang et al., 2002; Doi etal., 2003; Myers et al., 2003). siRNAs result when transposons, virusesor endogenous genes express long dsRNA or when dsRNA is introducedexperimentally into plant or animal cells to trigger gene silencing, aprocess known as RNA interference (RNAi) (Fire et al., 1998; Hamiltonand Baulcombe, 1999; Zamore et al., 2000; Elbashir et al., 2001a;Hammond et al., 2001; Sijen et al., 2001; Catalanotto et al., 2002). Incontrast, miRNAs are the products of endogenous, non-coding genes whoseprecursor RNA transcripts can form small stem-loops from which maturemiRNAs are cleaved by Dicer (Lagos-Quintana et al., 2001; Lau et al.,2001; Lee and Ambros, 2001; Lagos-Quintana et al., 2002; Mourelatos etal., 2002; Reinhart et al., 2002; Ambros et al., 2003; Brennecke et al.,2003; Lagos-Quintana et al., 2003; Lim et al., 2003a; Lim et al.,2003b). miRNAs are encoded by genes distinct from the mRNAs whoseexpression they control.

siRNAs were first identified as the specificity determinants of the RNAinterference (RNAi) pathway (Hamilton and Baulcombe, 1999; Hammond etal., 2000), where they act as guides to direct endonucleolytic cleavageof their target RNAs (Zamore et al., 2000; Elbashir et al., 2001a).Prototypical siRNA duplexes are 21 nt, double-stranded RNAs that contain19 base pairs, with two-nucleotide, 3′ overhanging ends (Elbashir etal., 2001a; Nykänen et al., 2001; Tang et al., 2003). Active siRNAscontain 5′ phosphates and 3′ hydroxyls (Zamore et al., 2000; Boutla etal., 2001; Nykänen et al., 2001; Chiu and Rana, 2002). Similarly, miRNAscontain 5′ phosphate and 3′ hydroxyl groups, reflecting their productionby Dicer (Hutvàgner et al., 2001; Mallory et al., 2002).

In plants, miRNAs regulate the expression of developmentally importantproteins, often by directing mRNA cleavage (Rhoades et al., 2002;Reinhart et al., 2002; Llave et al., 2002a; Llave et al., 2002b; Xie etal., 2003; Kasschau et al., 2003; Tang et al., 2003; Chen, 2003).Whereas plant miRNA's show a high degree of complementarity to theirmRNA targets, animal miRNA's have only limited complementarity to themRNAs whose expression they control (Lee et al., 1993; Wightman et al.,1993; Olsen and Ambros, 1999; Reinhart et al., 2000; Slack et al., 2000;Abrahante et al., 2003; Brennecke et al., 2003; Lin et al., 2003; Xu etal., 2003). Animal miRNAs are thought to repress mRNA translation,rather than promote target mRNA destruction (Lee et al., 1993; Wrightmanet al., 1993; Olsen and Ambross, 1999; Brennecke et al., 2003). Recentevidence suggests that the two classes of small RNAs are functionallyinterchangeable, with the choice of mRNA cleavage or translationalrepression determined solely by the degree of complementarity betweenthe small RNA and its target (Schwarz and Zamore, 2002; Hutvàgner andZamore, 2002; Zeng et al., 2003; Doench et al., 2003). Furthermore,siRNAs and miRNAs are found in similar, if not identical complexes,suggesting that a single, bifunctional complex the RNA-induced silencingcomplex (RISC)-mediates both cleavage and translational control(Mourelatos et al., 2002; Hutvàgner and Zamore, 2002; Caudy et al.,2002; Martinez et al., 2002). Nonetheless, studies in both plants andanimals show that at steady-state, siRNAs and miRNAs differ in at leastone crucial respect: in vivo and in vitro, siRNAs are double-stranded,whereas miRNAs are single-stranded (Lee et al., 1993; Hamilton andBaulcombe, 1999; Pasquinelli et al., 2000; Reinhart et al., 2000;Elbashir et al., 2001a; Djikeng et al., 2001; Nykänen et al., 2001;Lagos-Quintana et al., 2001; Lau et al., 2001; Lee and Ambros, 2001;Lagos-Quintana et al., 2002; Reinhart et al., 2002; Llave et al., 2002a;Silhavy et al., 2002; Llave et al., 2002b; Tang et al., 2003).

siRNA duplexes can assemble into RISC in the absence of target mRNA,both in vivo and in vitro (Tuschl et al., 1999; Hammond et al., 2000;Zamore et al., 2000). Each RISC contains only one of the two strands ofthe siRNA duplex (Martinez et al., 2002). Since siRNA duplexes have noforeknowledge of which siRNA strand will guide target cleavage, bothstrands must assemble with the appropriate proteins to form a RISC.Previously, we and others showed that both siRNA strands are competentto direct RNAi (Tuschl et al., 1999; Hammond et al., 2000; Zamore etal., 2000; Elbashir et al., 2001b; Elbashir et al., 2001a; Nykänen etal., 2001). That is, the anti-sense strand of an siRNA can directcleavage of a corresponding sense RNA target, whereas the sense siRNAstrand directs cleavage of an anti-sense target. In this way, siRNAduplexes appear to be functionally symmetric. The ability to controlwhich strand of an siRNA duplex enters into the RISC complex to directcleavage of a corresponding RNA target would provide a significantadvance for both research and therapeutic applications of RNAitechnology.

SUMMARY OF THE INVENTION

A key step in RNA interference (RNAi) is the assembly of a catalyticallyactive protein-RNA complex, the RNA-induced silencing complex (RISC),that mediates target RNA cleavage. The instant invention is based, atleast in part, on the discovery that the two strands of a siRNA duplexdo not contribute equally to RISC assembly. Rather, both the absoluteand the relative stabilites of the base pairs at the 5′ ends of the twosiRNA strands determines the degree to which each strand participates inthe RNAi pathway. In fact, siRNA can be functionally asymmetric, withonly one of the two strands able to trigger RNAi. The present inventionis also based on the discovery that single stranded miRNAs are initiallygenerated as siRNA-like duplexes whose structures predestine one strandto enter the RISC and the other strand to be destroyed. This findinghelps to explain the biogenesis of single-stranded miRNAs; the miRNAstrand of a short-lived, siRNA duplex-like intermediate is assembledinto a RISC complex, causing miRNAs to accumulate in vivo assingle-stranded RNAs.

The present invention is further based on the discovery that RISC cancleave RNA targets with up to five contiguous mismatches at the siRNA 5′end and eight mismatches at the siRNA 3′ end, indicating that 5′ basescontribute disproportionately to target RNA binding, but do not play arole in determining the catalytic rate, kcat. This finding explains howthe 5′, central and 3′ sequences of the siRNA guide strand function todirect target cleavage.

The invention is further based on the discovery that the 3′ bases of thesiRNA contribute much less than 5′ bases to the overall strength ofbinding, but instead help to establish the helical geometry required forRISC-mediated target cleavage, consistent with the view that catalysisby RISC requires a central A-form helix (Chiu et al., 2003). Thisfinding indicates that complementarity is essential for translationalrepression by siRNAs designed to act like animal miRNAs, which typicallyrepress translation (Doench et al., 2004).

The present invention is further based on the discovery that when ansiRNA fails to pair with the first three, four or five nucleotides ofthe target RNA, the phosphodiester bond severed in the target RNA isunchanged; for perfectly matched siRNA, RISC measures the site ofcleavage from the siRNA 5′ end (Elbashir et al., 2001; Elbashir et al.,2001). This finding indicates that the identity of the scissilephosphate is determined prior to the encounter of the RISC with itstarget RNA, perhaps because the RISC endonuclease is positioned withrespect to the siRNA 5′ end during RISC assembly.

Accordingly, the instant invention features methods of enhancing theefficacy and specificity of RNAi. Also provided is a method ofdecreasing silencing of an inadvertent target by an RNAi agent. Theinvention further features compositions, including siRNAs, shRNAs, aswell as vectors and transgenes, for mediating RNAi. The RNAi agents ofthe invention have improved specificity and efficacy in mediatingsilencing of a target gene.

The design rules for enhancing the efficacy and specificity of RNAiagents can also be applied to other RNA silencing agents, in particularagents that mediate translational repression of a target gene. Suchsilencing agents include, for example, siRNA-like duplexes that includea miRNA strand, as described supra. Methods featuring such silencingagents are also described herein.

Other features and advantages of the invention will be apparent from thefollowing detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. RNAi mediated by asymmetric duplex and single-stranded siRNAs.(A) Schematic showing relevant portions of the sense and anti-sensetarget RNA sequences (SEQ ID NOS 1 & 2). (B) Schematic showing siRNAduplex sequence (SEQ ID NOS 3 & 4) and graph depicting RNAi mediated bythe antisense and sense strands. (C) Schematic showing siRNA sequencesof individual single strands (SEQ ID NOS 3 & 4) and graph depicting RNAimediated by the single strands. (D) Bar graph depicting fraction oftotal siRNA present as single-strand. (E) Schematic showing siRNA duplexsequence (SEQ ID NOS 5 & 4) containing G:U wobble base pair and graphdepicting RNAi mediated by the antisense and sense strands.

FIG. 2. RNAi mediated by asymmetric duplex siRNAs. (A) Schematic showingrelevant portions of the sense and anti-sense target RNA sequences (SEQID NOS 6 & 7). (B) Schematic showing siRNA duplex sequence (bases 13-33of SEQ ID NO: 1 & SEQ ID NO: 8) and graph depicting RNAi mediated by theantisense and sense strands. (C) Schematic showing siRNA duplex sequence(SEQ ID NOS 9 & 10) containing A:U mismatch and graph depicting RNAimediated by the antisense and sense strands. (D) Schematic showing siRNAduplex sequence (SEQ ID NOS 9 & 8) containing G:U mismatch and graphdepicting RNAi mediated by the antisense and sense strands. (E)Schematic showing siRNA duplex sequence (bases 13-33 of SEQ ID NO: 1 &SEQ ID NO: 10) containing C:A mismatch and graph depicting RNAi mediatedby the antisense and sense strands.

FIG. 3. RNAi mediated by asymmetric duplex siRNAs. (A) Schematic showingrelevant portions of the sense and anti-sense target RNA sequences (SEQID NOS 11 & 12). (B) Schematic showing siRNA duplex sequence (SEQ ID NOS13 & 14) and graph depicting RNAi mediated by the antisense and sensestrands. (C) Schematic showing siRNA duplex sequence (SEQ ID NOS 15 &14) containing A:G mismatch and graph depicting RNAi mediated by theantisense and sense strands. (D) Schematic showing siRNA duplex sequence(SEQ ID NOS 13 & 16) containing C:U mismatch and graph depicting RNAimediated by the antisense and sense strands. (E) Schematic showing siRNAduplex sequence (SEQ ID NOS 15 & 16) containing A:U base pair and graphdepicting RNAi mediated by the antisense and sense strands. (F)Schematic showing siRNA duplex sequence (SEQ ID NOS 13 & 17) containingA:G mismatch and graph depicting RNAi mediated by the antisense andsense strands. (G) Schematic showing siRNA duplex sequence (SEQ ID NOS18 &14) containing C:U mismatch and graph depicting RNAi mediated by theantisense and sense strands. (H) Schematic showing siRNA duplex sequence(SEQ ID NOS 18 & 17) containing A:U base pair and graph depicting RNAimediated by the antisense and sense strands. (I) Schematic of individualsingle-strands of siRNAs (SEQ ID NOS 14, 16, 15, & 13 respectively inorder of appearance) and graph depicting RNAi mediated by the individualsingle-strands.

FIG. 4. RNAi mediated by asymmetric duplex siRNAs containing inosine.(A) Schematic showing siRNA duplex sequence (SEQ ID NOS 19 & 14) havinginosine at 5′ end of sense strand and graph depicting RNAi mediated bythe antisense and sense strands. (B) Schematic showing siRNA duplexsequence (SEQ ID NOS 13 & 20) having inosine at 5′ end of antisensestrand and graph depicting RNAi mediated by the antisense and sensestrands. (C) Schematic showing siRNA duplex sequence (SEQ ID NOS 19 &20) containing inosine in both strands and graph depicting RNAi mediatedby the antisense and sense strands. (D) Schematic showing individualsiRNA strands (SEQ ID NOS 19 & 20) containing inosine and graphdepicting RNAi mediated by the individual single-strands.

FIG. 5. Symmetric cleavage of pre-let-7 by Dicer. (A) Analysis ofcleavage products produced on 5′ side of precursor stem (let-7). (B)Analysis of cleavage products produced on 3′ side of precursor stem(let-7*). (C) Conceptual dicing of pre-let-7 (SEQ ID NO: 21) to adeduced pre-let-7 siRNA (SEQ ID NOS 22 & 23).

FIG. 6. Analysis of Drosphilia miRNA genes for predicted miRNA andmiRNA*. (A) Conceptual dicing of 26 published Drosphilia miRNA genes toa deduced duplex siRNA (SEQ ID NOS 24-72). (B) Amounts of miR-10 andmiR-10* detected in vivo.

FIG. 7. Schematic representing mechanism of RISC assembly from pre-miRNAor dsRNA.

FIG. 8. Reduction of off-target silencing by sense strand. (A) Sense andanti-sense sod1 target RNA sequences (SEQ ID NOS 73 & 74). (B) Schematicshowing siRNA duplex sequence (SEQ ID NOS 75 & 76) and graph depictingRNAi mediated by the antisense and sense strands. (C) Schematic showingsiRNA duplex sequence (SEQ ID NOS 75 & 77) containing G:U wobble basepair and graph depicting RNAi mediated by the antisense and sensestrands. (D) Schematic showing individual siRNA strands (SEQ ID NOS75-77) and graph depicting RNAi mediated by the individualsingle-strands. (E) Thermodynamic analysis of siRNA strand 5′ ends forthe siRNA duplex in (B). AG (kcal/mole) was calculated in 1M NaCl at 37°C.

FIG. 9. Enhancement of silencing by antisense strand. (A) Schematicshowing relevant portions of the sense (SEQ ID NO: 78) and anti-sense(SEQ ID NO: 79) target RNA sequences. (B) Schematic showing siRNA duplexsequence (SEQ ID NOS 80 & 81) and graph depicting RNAi mediated by theantisense and sense strands. (C) Schematic showing siRNA duplex sequence(SEQ ID NOS 82 & 83) containing A:U base pair and graph depicting RNAimediated by the antisense and sense strands. (D) Schematic showing siRNAduplex sequence (SEQ ID NOS 82 & 81) containing A:G mismatch and graphdepicting RNAi mediated by the antisense and sense strands. (E)Thermodynamic analysis of siRNA strand 5′ ends for the siRNA duplex in(B). AG (kcal/mole) was calculated in 1M NaCl at 37° C.

FIG. 10. The relative thermodynamic stability of the first four basepairs of the siRNA strands (SEQ ID NOS 3-5) explains siRNA functionalasymmetry. Thermodynamic analysis of siRNA strand 5′ ends for the siRNAsin FIGS. 1B and 1E. ΔG (kcal/mole) was calculated in 1M NaCl at 37° C.

FIG. 11. The first four base pairs of the siRNA duplex determinestrand-specific activity. Internal, single-nucleotide mismatches (A-F)(SEQ ID NOS 13, 14, & 84-89) near the 5′ ends of an siRNA strandgenerate functional asymmetry, but internal G:U wobble pairs (G-I) (SEQID NOS 13, 14, 90, & 91) do not.

FIG. 12. Increased rate of siRNA efficiency when duplexes have dTdTmismatched tails.

FIG. 13. Product release limits the rate of catalysis by RISC. (a) ATPstimulates multiple rounds of RISC cleavage of the RNA target. siRNA wasincubated with ATP in Drosophila embryo lysate, then NEM was added toquench RISC assembly and to disable the ATP-regenerating system. Theenergy regenerating system was either restored by adding additionalcreatine kinase (+ATP) or the reaction was ATP-depleted by addinghexokinase and glucose (ATP). The target RNA concentration was 49 nM andthe concentration of RISC was ˜4 nM. The siRNA sequence and its targetRNA is given in FIG. 21A. (b) In the absence of ATP, cleavage by RISCproduces a pre-steady state burst equal, within error, to theconcentration of active RISC. The target concentration was 110 nM andthe RISC concentration was ˜4 nM. (c) Catalysis by RISC is not enhancedby ATP under single-turnover conditions. RISC was present in ˜8-foldexcess over target. Each data point represents the average of twotrials.

FIG. 14. In the absence of ATP, mismatches between the 3′ end of thesiRNA guide strand and the target RNA facilitate product release, butreduce the rate of target cleavage. (a) Representative siRNA sequencesare shown aligned with the target sequence (SEQ ID NOS 92-101). ThesiRNA guide strand is in color (5′ to 3′) and the mismatch with thetarget site is highlighted in yellow. A complete list of siRNA sequencesappears in FIG. 21. (b) The steady-state rate of cleavage in thepresence and absence of ATP was determined for siRNAs with zero to four3′ mismatches with the target site. The target RNA concentration was 49nM and the concentration of RISC was either ˜4 nM (no mismatches) or ˜6nM (1 to 4 mismatches). The steady-state velocity with ATP, relative tothe velocity without ATP is shown for each siRNA. (c) Time course ofcleavage for perfectly matched (˜16-fold excess of RISC relative totarget) and mismatched (˜80-fold excess of RISC) siRNA. (d) Datarepresentative of those used in the analysis in (c) for target cleavagedirected by siRNAs with zero, four, and five 3′ mismatches.

FIG. 15. Remarkable tolerance of RISC for 3′ mismatches. (a) Eachadditional 3′ mismatch further reduced the rate of cleavage by RISC. Thesteady-state rates of cleavage were determined for siRNA with zero, one,two, and four mismatches under multiple-turnover conditions (˜49 nMtarget mRNA and ˜4-6 nM RISC). (b) Analysis of siRNAs bearing zero tofive 3′ mismatches with the target RNA under conditions of slight enzymeexcess (˜2-fold more RISC than target). siRNA sequences andcorresponding target RNA sequences used in (a) and (b) are shown in FIG.14A and FIG. 21A. (c) Extended endpoint analysis of RISC cleavage underconditions of ˜80-fold enzyme excess reveals that cleavage can occur forsiRNAs with as many as eight mismatches to the target RNA. Note thedifferent time scales in (c) versus (b). All reactions were understandard in vitro RNAi (+ATP) conditions. siRNA sequences andcorresponding target RNA sequences used in (c) are shown in FIG. 21B.

FIG. 16. Limited tolerance of RISC for 5′ mismatches. (a) RISC cleavagewas analyzed as in FIG. 21C using 5′ mismatched siRNAs, whose sequencesare given in FIG. 21. The target RNA was the same for all siRNAs. siRNAsequences and the corresponding target RNA sequence are shown in FIG.21E. (b) RISC cleavage was analyzed using a single siRNA sequence.Mismatches were created by altering the sequence of the target RNA. Forthe target containing compensatory mutations, the target concentrationwas 0.25 nM and the siRNA concentration was ˜20 nM; RISC concentrationwas not determined. The asterisk denotes a 15 second time-point. siRNAsequences and corresponding target RNA sequencse are shown in FIG. 21F.(c) RISC cleavage was analyzed by incubating 50 nM siRNA with 0.5 nMtarget RNA. 3′ mismatches were created by modifying the target sequence,and 5′ mismatches by changing the siRNA. Target and siRNA sequences aregiven in FIG. 21C. (d) Perfectly base-paired and 5′ mismatched siRNAsdirect cleavage at the same phosphodiester bond. Cleavage reactions wereperformed with ˜20 nM RISC generated from 50 nM siRNA and 0.5 nM targetRNA and analyzed on an 8% denaturing polyacrylamide sequencing gel. Thetarget mRNA was 182 nt and 5′ cleavage product was 148 nt. After RISCwas assembled, the extract was treated with NEM to inactivate nucleases(Schwartz et al., 2004). After NEM treatment, the ATP regeneratingsystem was restored by adding additional creatine kinase, then targetRNA was added and the incubation continued for the indicated time. OH—denotes a base hydrolysis ladder. The siRNA and corresponding target RNAsequences used in this experiment are shown in FIG. 21E.

FIG. 17. Michaelis-Menten and Ki analysis for matched and mismatchedsiRNAs reveal distinct contributions to binding and catalysis for the5′, central, and 3′ regions of the siRNA. (a) siRNA was assembled intoRISC under standard in vitro RNAi conditions, then diluted to achievethe desired RISC concentration. The initial rates of cleavage weredetermined for increasing concentrations of 5′ 32P-cap-radiolabeledtarget mRNA. Plot of initial velocity versus substrate concentration. KMand Vmax were determined by fitting the data to the Michaelis-Mentenequation. See Table 1 for analysis. Representative initial ratedeterminations appear in FIG. 20A, and the siRNA sequences used in thisexperiment are shown in FIGS. 21A and 21E. (b) Ki values were determinedin competition assays using 2′-O-methyl oligonucleotides bearing 5′,central, and 3′ mismatches to the siRNA guide strand. Representativedata are presented in FIG. 20B, and a complete list of the 2′-O-methyloligonucleotides used appears in FIG. 21D.

FIG. 18. A model for the cycle of RISC assembly, target recognition,catalysis, and recycling.

FIG. 19. Exogenously programmed RISC is a bona fide enzyme siRNA wasassembled into RISC for 1 hour in a standard in vitro RNAi reaction,then assembly was quenched with N-ethyl maleimide (NEM)21,29. The amountof RISC formed was determined by measuring 32P-radiolabeled siRNAretained on a tethered 5′-biotinylated, 31-nt, 2′-O-methyloligonucleotide complementary to the guide strand of the siRNA. RISCbinds essentially irreversibly to tethered 2′-O-methyl oligonucleotides,but cannot cleave these RNA-analogs (Hutvàgner et al., 2004; Schwartz etal., 2003). In all experiments, target-cleaving activity was notdetected in the supernatant, demonstrating that all the active RISC wasretained on the beads. (a) Sequence of the siRNA used (SEQ ID NOS 93 &94) (guide strand in red, 32P-radiolabel marked with an asterisk).Drosophila let-7 is not expressed in 0-2 hour embryos (Hutvàgner et al.,2001), so the only source of let-7 in the in vitro reactions was theexogenous let-7 siRNA. The 5′ end of the guide strand of the let-7 siRNAis predicted to be thermodynamically more stable than the 5′ end of thepassenger strand, explaining why only a low concentrations oflet-7-programmed RISC is formed (Schwartz et al., 2003, Khvorova et al.,2003). The maximum amount of RISC assembled varies widely with siRNAsequence. The siRNAs used in FIGS. 3-8 were designed to load=5-fold moreguide strand-containing RISC (Hutvàgner et al., 2001; Schwartz et al.,2003) (b) Representative gel confirming that the RISC was removed by thetethered 2′-O-methyl oligonucleotide. A reaction prior to incubationwith the tethered 2′-O-methyl oligonucleotide (pre) was compared to thesupernatant of a reaction incubated with beads alone (mock), and thesupernatant of a reaction incubated with the complementary tethered2′-O-methyl oligonucleotide (post). The buffer reaction contained nosiRNA. (c) Analysis of the amount of RISC assembled at various siRNAconcentrations. 5′ 32P-radiolabeled siRNA was incubated with lysate for1 hour, then reactions were quenched by treatment with NEM, and RISCconcentration was measured using the tethered 2′-O-methyloligonucleotide method.

FIG. 20. Michaelis-Menton and Competitor Analysis of RISC (a)Representative data for the determination of initial velocities for theperfectly matched siRNA. Black, 1 nM target; red, 5 nM; blue, 20 nM; andgreen, 60 nM. (b) Three independent experiments for inhibition by afully complementary 2′-O-methyl oligonucleotide competitor. ˜1 nM RISCand 5 nM 32P-cap-radiolabeled target mRNA were incubated with increasingconcentration of competitor, and the initial velocities were calculatedand plotted versus competitor concentration.

FIG. 21. siRNAs, target sites, and 2′-O-methyl oligonucleotides (SEQ IDNOS 92-143) used in this study. (a) List of siRNA and correspondingtarget RNA sequences used in the experiments described in Table 1 andFIGS. 14B-D, 15A, 15B, 17A, and 20A. (b) List of siRNA and correspondingtarget RNA sequences used in the experiment described in FIG. 15C. (c)List of siRNA and corresponding target RNA sequences used in theexperiment described in FIG. 16C. (d) List of siRNAs and correspondingtarget 2′-O-methyl oligonucleotide sequences used in the experimentsdescribed in FIGS. 17B and 20B. (e) List of siRNA and correspondingtarget RNA sequences used in the experiments described in FIGS. 16A,16D, 17A, and Table 1. (f) List of siRNA and corresponding target RNAsequences used in the experiment described in FIG. 16B.

DETAILED DESCRIPTION OF THE INVENTION

A key step in RNA interference (RNAi) is the assembly of a catalyticallyactive protein-RNA complex, the RNA-induced silencing complex (RISC),that mediates target RNA cleavage. Each RISC contains one of the twostrands of the small interfering RNA (siRNA) duplex that triggers RNAi.The instant invention is based, at least in part, on the discovery thatthe two siRNA strands do not contribute equally to RISC assembly. Smallchanges in siRNA sequence were found to have profound and predictableeffects on the extent to which the two strands of an siRNA duplex enterthe RNAi pathway, a phenomenon termed siRNA functional “asymmetry”. Thediscoveries described herein reveal that the strength of thebase-pairing interactions made by the 5′ end of each siRNA strand withthe 3′ region of strand to which it is paired determines which of thetwo strands participates in the RNAi pathway. RISC assembly appears tobe governed by an enzyme that initiates unwinding of an siRNA duplex atthe siRNA strand whose 5′ end is less tightly paired to thecomplementary siRNA strand.

Remarkably, such highly asymmetric siRNA duplexes resemble proposedintermediates in the biogenesis pathway of microRNA (miRNA) (Hutvàgnerand Zamore, 2002; Reinhart et al., 2002; Lim et al., 2003b). miRNAs areendogenous, ˜21-nt single-stranded RNAs processed by Dicer fromstem-loop RNA precursors that regulate gene expression in animals andplants. A striking feature of miRNA precursors is their lack of fullcomplementarity in the stem region. The discoveries presented hereinindicate an important role for the discontinuities in the stem region ofmiRNAs; it is likely that miRNAs are initially generated from theirprecursor RNAs as siRNA-like duplexes, and that the structure of theseduplexes predestines the miRNA strand to enter the RISC and the otherstrand to be destroyed. Thus, nature appears to have optimized the stemportion of miRNAs to follow a set of rules dictating which strand entersthe RISC complex.

The discoveries made by the instant inventors provide rules according towhich siRNAs and shRNAs can be designed that are fully asymmetric, withonly one of the two siRNA strands competent to enter the RISC complex.By applying these rules to the selection and design of a targeted RNAiagent, e.g., siRNAs and shRNAs, the antisense strand of the RNAi agentcan be predictably directed to enter the RISC complex and mediate targetRNA cleavage. Similarly, the sense strand can be discouraged fromentering the RISC complex, thereby reducing or eliminating undesiredsilencing of an inadvertent target by the sense strand.

Accordingly, the instant invention provides methods for improving theefficiency (or specificity) of an RNAi reaction comprising identifyingan off target RNAi activity mediated by the sense strand of an RNAiagent, and modifying the RNAi agent such that the base pair strengthbetween the 5′ end of the antisense strand and the 3′ end of the sensestrand is lessened relative to the base pair strength of the 5′ end ofthe sense strand and the 3′ end of the antisense strand (e.g., relativeto the premodified RNAi agent), such that the sense strand is lesseffective at entering RISC (e.g., less effective than the premodifiedRNAi agent).

The instant invention also provides methods for improving the efficiency(or specificity) of an RNAi reaction comprising modifying (e.g.,increasing) the asymmetry of the RNAi agent such that the ability of thesense or second strand to mediate RNAi (e.g., mediate cleavage of targetRNA) is lessened. In preferred embodiments, the asymmetry is increasedin favor of the 5′ end of the first strand, e.g., lessening the bondstrength (e.g., the strength of the interaction) between the 5′ end ofthe first strand and 3′ end of the second strand relative to the bondstrength (e.g., the strength of the interaction) between the 5′ end ofthe second strand and the 3′ end of the first strand. In otherembodiments, the asymmetry is increased in favor of the 5′ end of thefirst strand by increasing bond strength (e.g., the strength of theinteraction) between the 5′ end of the second or sense strand and the 3′end of the first or antisense strand, relative to the bond strength(e.g., the strength of the interaction) between the 5′ end of the firstand the 3′ end of the second strand. In embodiments of the invention,the bond strength is increased, e.g., the H bonding is increased betweennucleotides or analogs at the 5′ end, e.g., within 5 nucleotides of thesecond or sense strand (numbered from the 5′ end of the second strand)and complementary nucleotides of the first or antisense strand. It isunderstood that the asymmetry can be zero (i.e., no asymmetry), forexample, when the bonds or base pairs between the 5′ and 3′ terminalbases are of the same nature, strength or structure. More routinely,however, there exists some asymmetry due to the different nature,strength or structure of at least one nucleotide (often one or morenucleotides) between terminal nucleotides or nucleotide analogs.

Accordingly, in one aspect, the instant invention provides a method ofenhancing the ability of a first strand of a RNAi agent to act as aguide strand in mediating RNAi, involving lessening the base pairstrength between the 5′ end of the first strand and the 3′ end of asecond strand of the duplex as compared to the base pair strengthbetween the 3′ end of the first strand and the 5′ end of the secondstrand.

In a related aspect, the invention provides a method of enhancing theefficacy of a siRNA duplex, the siRNA duplex comprising a sense and anantisense strand, involving lessening the base pair strength between theantisense strand 5′ end (AS 5′) and the sense strand 3′ end (S 3′) ascompared to the base pair strength between the antisense strand 3′ end(AS 3′) and the sense strand 5′ end (S 5′), such that efficacy isenhanced.

In another aspect of the invention, a method is provided for promotingentry of a desired strand of an siRNA duplex into a RISC complex,comprising enhancing the asymmetry of the siRNA duplex, such that entryof the desired strand is promoted. In one embodiment of this aspect ofthe invention, the asymmetry is enhanced by lessening the base pairstrength between the 5′ end of the desired strand and the 3′ end of acomplementary strand of the duplex as compared to the base pair strengthbetween the 3′ end of the desired strand and the 5′ end of thecomplementary strand.

In another aspect of the invention, a siRNA duplex is providedcomprising a sense strand and an antisense strand, wherein the base pairstrength between the antisense strand 5′ end (AS 5′) and the sensestrand 3′ end (S 3′) is less than the base pair strength between theantisense strand 3′ end (AS 3′) and the sense strand 5′ end (S 5′), suchthat the antisense strand preferentially guides cleavage of a targetmRNA.

In one embodiment of these aspects of the invention, the base-pairstrength is less due to fewer G:C base pairs between the 5′ end of thefirst or antisense strand and the 3′ end of the second or sense strandthan between the 3′ end of the first or antisense strand and the 5′ endof the second or sense strand.

In another embodiment, the base pair strength is less due to at leastone mismatched base pair between the 5′ end of the first or antisensestrand and the 3′ end of the second or sense strand. Preferably, themismatched base pair is selected from the group consisting of G:A, C:A,C:U, G:G, A:A, C:C and U:U.

In one embodiment, the base pair strength is less due to at least onewobble base pair, e.g., G:U, between the 5′ end of the first orantisense strand and the 3′ end of the second or sense strand.

In another embodiment, the base pair strength is less due to at leastone base pair comprising a rare nucleotide, e.g, inosine (I).Preferably, the base pair is selected from the group consisting of anI:A, I:U and I:C.

In yet another embodiment, the base pair strength is less due to atleast one base pair comprising a modified nucleotide. In preferredembodiments, the modified nucleotide is selected from the groupconsisting of 2-amino-G, 2-amino-A, 2,6-diamino-G, and 2,6-diamino-A.

In several embodiments of these aspects of the invention, the RNAi agentis a siRNA duplex or is derived from an engineered precursor, and can bechemically synthesized or enzymatically synthesized.

In another aspect of the instant invention, compositions are providedcomprising a siRNA duplex of the invention formulated to facilitateentry of the siRNA duplex into a cell. Also provided are pharmaceuticalcomposition comprising a siRNA duplex of the invention.

Further provided are an engineered pre-miRNA comprising a siRNA duplexas described above, as well as a vector encoding the pre-miRNA. Inrelated aspects, the invention provides a pri-miRNA comprising thepre-miRNA, as well as a vector encoding the pri-miRNA.

Also featured in the instant invention are small hairpin RNA (shRNA)comprising nucleotide sequence identical to the sense and antisensestrand of the siRNA duplexes as described above. In one embodiment, thenucleotide sequence identical to the sense strand is upstream of thenucleotide sequence identical to the antisense strand. In anotherembodiment, the nucleotide sequence identical to the antisense strand isupstream of the nucleotide sequence identical to the sense strand.Further provided are vectors and transgenes encoding the shRNAs of theinvention.

In yet another aspect, the invention provides cells comprising thevectors featured in the instant invention. Preferably, the cell is amammalian cell, e.g., a human cell.

In other aspects of the invention, methods of enhancing silencing of atarget mRNA, comprising contacting a cell having an RNAi pathway with aRNAi agent as described above under conditions such that silencing isenhanced.

Also provided are methods of enhancing silencing of a target mRNA in asubject, comprising administering to the subject a pharmaceuticalcomposition comprising a RNAi agent as described above such thatsilencing is enhanced.

Further provided is a method of decreasing silencing of an inadvertenttarget mRNA by a dsRNAi agent, the dsRNAi agent comprising a sensestrand and an antisense strand involving the steps of: (a) detecting asignificant degree of complementarity between the sense strand and theinadvertent target; and (b) enhancing the base pair strength between the5′ end of the sense strand and the 3′ end of the antisense strandrelative to the base pair strength between the 3′ end of the sensestrand and the 5′ end of the antisense strand; such that silencing ofthe inadvertent target mRNA is decreased. In a preferred embodiment, thesilencing of the inadvertent target mRNA is decreased relative tosilencing of a desired target mRNA.

The design rules described supra can also be applied to other classes ofRNA silencing agents, for example, siRNA-like duplexes that include amiRNA strand. In particular, the design rules can be applied to suchsiRNA-like duplexes to promote entry of the desired strand of the duplex(e.g., the miRNA strand) into a RISC complex. The degree ofcomplementarity between the miRNA strand and its target determineswhether the small RNA mediates mRNA cleavage or translationalrepression, as described supra. Such duplexes are useful for effectivelydelivering miRNAs, for example, to cells or animals for therapeuticpurposes.

Accordingly, in one aspect, the instant invention provides a method ofenhancing the ability of a first strand of a siRNA-like duplex to act inmediating RNA silencing (e.g., RNAi or translational repression),involving lessening the base pair strength between the 5′ end of thefirst strand and the 3′ end of a second strand of the duplex as comparedto the base pair strength between the 3′ end of the first strand and the5′ end of the second strand.

In a related aspect, the invention provides a method of enhancing theefficacy of a siRNA-like duplex, the siRNA-like duplex comprising amiRNA and an miRNA* strand, involving lessening the base pair strengthbetween the miRNA strand 5′ end (miRNA 5′) and the miRNA* strand 3′ end(miRNA* 3′) as compared to the base pair strength between the miRNAstrand 3′ end (miRNA 3′) and the miRNA* strand 5′ end (miRNA* 5′), suchthat efficacy is enhanced.

In another aspect of the invention, a method is provided for promotingentry of a desired strand of an siRNA-like duplex into a RISC complex,comprising enhancing the asymmetry of the siRNA duplex, such that entryof the desired strand is promoted. In one embodiment of this aspect ofthe invention, the asymmetry is enhanced by lessening the base pairstrength between the 5′ end of the desired strand and the 3′ end of acomplementary strand of the duplex as compared to the base pair strengthbetween the 3′ end of the desired strand and the 5′ end of thecomplementary strand.

In another aspect of the invention, a siRNA-like duplex is providedcomprising a miRNA strand and an miRNA* strand, wherein the base pairstrength between the miRNA strand 5′ end (miRNA 5′) and the miRNA*strand 3′ end (miRNA* 3′) is less than the base pair strength betweenthe miRNA strand 3′ end (miRNA 3′) and the miRNA* strand 5′ end (miRNA*5′), such that the miRNA strand preferentially silences (e.g., cleavesor represses) a target mRNA.

In one embodiment of these aspects of the invention, the base-pairstrength is less due to fewer G:C base pairs between the 5′ end of thefirst or miRNA strand and the 3′ end of the second or miRNA* strand thanbetween the 3′ end of the first or miRNA strand and the 5′ end of thesecond or miRNA* strand.

In another embodiment, the base pair strength is less due to at leastone mismatched base pair between the 5′ end of the first or miRNA strandand the 3′ end of the second or miRNA* strand. Preferably, themismatched base pair is selected from the group consisting of G:A, C:A,C:U, G:G, A:A, C:C and U:U.

In one embodiment, the base pair strength is less due to at least onewobble base pair, e.g., G:U, between the 5′ end of the first or miRNAstrand and the 3′ end of the second or miRNA* strand.

In another embodiment, the base pair strength is less due to at leastone base pair comprising a rare nucleotide, e.g, inosine (I).Preferably, the base pair is selected from the group consisting of anI:A, I:U and I:C.

In yet another embodiment, the base pair strength is less due to atleast one base pair comprising a modified nucleotide. In preferredembodiments, the modified nucleotide is selected from the groupconsisting of 2-amino-G, 2-amino-A, 2,6-diamino-G, and 2,6-diamino-A.

In several embodiments of these aspects of the invention, the RNAsilencing agent is a siRNA-like duplex or is derived from an engineeredprecursor, and can be chemically synthesized or enzymaticallysynthesized.

In another aspect of the instant invention, compositions are providedcomprising a siRNA-like duplex of the invention formulated to facilitateentry of the siRNA-like duplex into a cell. Also provided arepharmaceutical composition comprising a siRNA-like duplex of theinvention.

Further provided are an engineered pre-miRNA comprising the siRNA-likeduplex as described above, as well as a vector encoding the pre-miRNA.In related aspects, the invention provides a pri-miRNA comprising thepre-miRNA, as well as a vector encoding the pri-miRNA.

Also featured in the instant invention are small hairpin RNA (shRNA)comprising nucleotide sequence identical to the miRNA and miRNA* strandof the siRNA-like duplex as described above. In one embodiment, thenucleotide sequence identical to the miRNA strand is upstream of thenucleotide sequence identical to the miRNA* strand. In anotherembodiment, the nucleotide sequence identical to the miRNA* strand isupstream of the nucleotide sequence identical to the miRNA strand.Further provided are vectors and transgenes encoding the shRNAs of theinvention.

In yet another aspect, the invention provides cells comprising thevectors featured in the instant invention. Preferably, the cell is amammalian cell, e.g., a human cell.

In other aspects of the invention, methods of enhancing silencing of atarget mRNA, comprising contacting a cell having an RNA silencingpathway with the RNA silencing agent as described above under conditionssuch that silencing is enhanced.

Also provided are methods of enhancing silencing of a target mRNA in asubject, comprising administering to the subject a pharmaceuticalcomposition comprising the RNA silencing agent as described above suchthat silencing is enhanced.

Further provided is a method of decreasing silencing of an inadvertenttarget mRNA by a dsRNA silencing agent, the dsRNA silencing agent (e.g.,a siRNA-like duplex) comprising a miRNA strand and a miRNA* strandinvolving the steps of: (a) detecting a significant degree ofcomplementarity between the miRNA* strand and the inadvertent target;and (b) enhancing the base pair strength between the 5′ end of themiRNA* strand and the 3′ end of the miRNA strand relative to the basepair strength between the 3′ end of the miRNA* strand and the 5′ end ofthe miRNA strand; such that silencing of the inadvertent target mRNA isdecreased. In a preferred embodiment, the silencing of the inadvertenttarget mRNA is decreased relative to silencing of a desired target mRNA.

So that the invention may be more readily understood, certain terms arefirst defined.

The term “nucleoside” refers to a molecule having a purine or pyrimidinebase covalently linked to a ribose or deoxyribose sugar. Exemplarynucleosides include adenosine, guanosine, cytidine, uridine andthymidine. The term “nucleotide” refers to a nucleoside having one ormore phosphate groups joined in ester linkages to the sugar moiety.Exemplary nucleotides include nucleoside monophosphates, diphosphatesand triphosphates. The terms “polynucleotide” and “nucleic acidmolecule” are used interchangeably herein and refer to a polymer ofnucleotides joined together by a phosphodiester linkage between 5′ and3′ carbon atoms.

The term “RNA” or “RNA molecule” or “ribonucleic acid molecule” refersto a polymer of ribonucleotides. The term “DNA” or “DNA molecule” or“deoxyribonucleic acid molecule” refers to a polymer ofdeoxyribonucleotides. DNA and RNA can be synthesized naturally (e.g., byDNA replication or transcription of DNA, respectively). RNA can bepost-transcriptionally modified. DNA and RNA can also be chemicallysynthesized. DNA and RNA can be single-stranded (i.e., ssRNA and ssDNA,respectively) or multi-stranded (e.g., double stranded, i.e., dsRNA anddsDNA, respectively). “mRNA” or “messenger RNA” is single-stranded RNAthat specifies the amino acid sequence of one or more polypeptidechains. This information is translated during protein synthesis whenribosomes bind to the mRNA.

As used herein, the term “RNA silencing agent” refers to an RNA which iscapable of preventing complete processing (e.g, the full translationand/or expression) of a mRNA molecule through a post-transcriptionalsilencing mechanism. RNA silencing agents include small (<50 b.p.),noncoding RNA molecules, for example RNA duplexes comprising pairedstrands, as well as precursor RNAs from which such small non-coding RNAscan be generated. Exemplary RNA silencing agents include siRNAs, miRNAs,and siRNA-like duplexes, as well as precursors thereof.

As used herein, the term “small interfering RNA” (“siRNA”) (alsoreferred to in the art as “short interfering RNAs”) refers to an RNA (orRNA analog) comprising between about 10-50 nucleotides (or nucleotideanalogs) which is capable of directing or mediating RNA interference.Preferably, an siRNA comprises between about 15-30 nucleotides ornucleotide analogs, more preferably between about 16-25 nucleotides (ornucleotide analogs), even more preferably between about 18-23nucleotides (or nucleotide analogs), and even more preferably betweenabout 19-22 nucleotides (or nucleotide analogs) (e.g., 19, 20, 21 or 22nucleotides or nucleotide analogs).

As used herein, the term “microRNA” (“miRNA”), also referred to in theart as “small temporal RNAs” (“stRNAs”), refers to a small (10-50nucleotide) RNA which is capable of directed or mediating RNA silencing.A “natural miRNA” refers to a microRNA that occurs naturally. An “miRNAdisorder” shall refer to a disease or disorder characterized by aaberrant expression or activity of a natural miRNA.

As used herein, the term “rare nucleotide” refers to a naturallyoccurring nucleotide that occurs infrequently, including naturallyoccurring deoxyribonucleotides or ribonucleotides that occurinfrequently, e.g., a naturally occurring ribonucleotide that is notguanosine, adenosine, cytosine, or uridine. Examples of rare nucleotidesinclude, but are not limited to, inosine, 1-methyl inosine,pseudouridine, 5,6-dihydrouridine, ribothymidine, ²N-methylguanosine and^(2,2)N,N-dimethylguanosine.

The term “nucleotide analog” or “altered nucleotide” or “modifiednucleotide” refers to a non-standard nucleotide, including non-naturallyoccurring ribonucleotides or deoxyribonucleotides. Preferred nucleotideanalogs are modified at any position so as to alter certain chemicalproperties of the nucleotide yet retain the ability of the nucleotideanalog to perform its intended function. Examples of preferred modifiednucleotides include, but are not limited to, 2-amino-guanosine,2-amino-adenosine, 2,6-diamino-guanosine and 2,6-diamino-adenosine.Examples of positions of the nucleotide which may be derivatized includethe 5 position, e.g., 5-(2-amino)propyl uridine, 5-bromo uridine,5-propyne uridine, 5-propenyl uridine, etc.; the 6 position, e.g.,6-(2-amino)propyl uridine; the 8-position for adenosine and/orguanosines, e.g., 8-bromo guanosine, 8-chloro guanosine,8-fluoroguanosine, etc. Nucleotide analogs also include deazanucleotides, e.g., 7-deaza-adenosine; O- and N-modified (e.g.,alkylated, e.g., N6-methyl adenosine, or as otherwise known in the art)nucleotides; and other heterocyclically modified nucleotide analogs suchas those described in Herdewijn, Antisense Nucleic Acid Drug Dev., 2000Aug. 10(4):297-310.

Nucleotide analogs may also comprise modifications to the sugar portionof the nucleotides. For example the 2′ OH-group may be replaced by agroup selected from H, OR, R, F, Cl, Br, I, SH, SR, NH₂, NHR, NR₂, COOR,or OR, wherein R is substituted or unsubstituted C₁-C₆ alkyl, alkenyl,alkynyl, aryl, etc. Other possible modifications include those describedin U.S. Pat. Nos. 5,858,988, and 6,291,438.

The phosphate group of the nucleotide may also be modified, e.g., bysubstituting one or more of the oxygens of the phosphate group withsulfur (e.g., phosphorothioates), or by making other substitutions whichallow the nucleotide to perform its intended function such as describedin, for example, Eckstein, Antisense Nucleic Acid Drug Dev. 2000 Apr.10(2):117-21, Rusckowski et al. Antisense Nucleic Acid Drug Dev. 2000Oct. 10(5):333-45, Stein, Antisense Nucleic Acid Drug Dev. 2001 Oct.11(5): 317-25, Vorobjev et al. Antisense Nucleic Acid Drug Dev. 2001Apr. 11(2):77-85, and U.S. Pat. No. 5,684,143. Certain of theabove-referenced modifications (e.g., phosphate group modifications)preferably decrease the rate of hydrolysis of, for example,polynucleotides comprising said analogs in vivo or in vitro.

The term “oligonucleotide” refers to a short polymer of nucleotidesand/or nucleotide analogs. The term “RNA analog” refers to anpolynucleotide (e.g., a chemically synthesized polynucleotide) having atleast one altered or modified nucleotide as compared to a correspondingunaltered or unmodified RNA but retaining the same or similar nature orfunction as the corresponding unaltered or unmodified RNA. As discussedabove, the oligonucleotides may be linked with linkages which result ina lower rate of hydrolysis of the RNA analog as compared to an RNAmolecule with phosphodiester linkages. For example, the nucleotides ofthe analog may comprise methylenediol, ethylene diol, oxymethylthio,oxyethylthio, oxycarbonyloxy, phosphorodiamidate, phosphoroamidate,and/or phosphorothioate linkages. Preferred RNA analogues include sugar-and/or backbone-modified ribonucleotides and/or deoxyribonucleotides.Such alterations or modifications can further include addition ofnon-nucleotide material, such as to the end(s) of the RNA or internally(at one or more nucleotides of the RNA). An RNA analog need only besufficiently similar to natural RNA that it has the ability to mediate(mediates) RNA interference.

As used herein, the term “RNA interference” (“RNAi”) (also referred toin the art as “gene silencing” and/or “target silencing”, e.g., “targetmRNA silencing”) refers to a selective intracellular degradation of RNA.RNAi occurs in cells naturally to remove foreign RNAs (e.g., viralRNAs). Natural RNAi proceeds via fragments cleaved from free dsRNA whichdirect the degradative mechanism to other similar RNA sequences. As usedherein, the term “translational repression” refers to a selectiveinhibition of mRNA translation. Natural translational repressionproceeds via miRNAs cleaved from shRNA precursors. Both RNAi andtranslational repression are mediated by RISC. Both RNAi andtranslational repression occur naturally or can be initiated by the handof man, for example, to silence the expression of target genes.

As used herein, the term “antisense strand” of an siRNA or RNAi agentrefers to a strand that is substantially complementary to a section ofabout 10-50 nucleotides, e.g., about 15-30, 16-25, 18-23 or 19-22nucleotides of the mRNA of the gene targeted for silencing. Theantisense strand or first strand has sequence sufficiently complementaryto the desired target mRNA sequence to direct target-specific RNAinterference (RNAi), e.g., complementarity sufficient to trigger thedestruction of the desired target mRNA by the RNAi machinery or process(RNAi interference) or complementarity sufficient to triggertranslational repression of the desired target mRNA. The term “sensestrand” or “second strand” of an siRNA or RNAi agent refers to a strandthat is complementary to the antisense strand or first strand. Antisenseand sense strands can also be referred to as first or second strands,the first or second strand having complementarity to the target sequenceand the respective second or first strand having complementarity to saidfirst or second strand. miRNA duplex intermediates or siRNA-likeduplexes include a miRNA strand having sufficient complementarity to asection of about 10-50 nucleotides of the mRNA of the gene targeted forsilencing and a miRNA* strand having sufficient complementarity to forma duplex with the miRNA strand.

As used herein, the term “guide strand” refers to a strand of an RNAiagent, e.g., an antisense strand of an siRNA duplex, that enters intothe RISC complex and directs cleavage of the target mRNA.

A “target gene” is a gene whose expression is to be selectivelyinhibited or “silenced.” This silencing can be achieved by cleaving themRNA of the target gene or via translational repression of the targetgene.

The term “engineered,” as in an engineered RNA precursor, or anengineered nucleic acid molecule, indicates that the precursor ormolecule is not found in nature, in that all or a portion of the nucleicacid sequence of the precursor or molecule is created or selected byman. Once created or selected, the sequence can be replicated,translated, transcribed, or otherwise processed by mechanisms within acell. Thus, an RNA precursor produced within a cell from a transgenethat includes an engineered nucleic acid molecule is an engineered RNAprecursor.

As used herein, the term “asymmetry”, as in the asymmetry of a RNAduplex (e.g. a siRNA duplex or siRNA-like or miRNA duplex), refers to aninequality of bond strength or base pairing strength between the duplextermini (e.g., between terminal nucleotides on a first strand andterminal nucleotides on an opposing second strand), such that the 5′ endof one strand of the duplex is more frequently in a transient unpaired,e.g, single-stranded, state than the 5′ end of the complementary strand.This structural difference determines that one strand of the duplex ispreferentially incorporated into a RISC complex. The strand whose 5′ endis less tightly paired to the complementary strand will preferentiallybe incorporated into RISC and mediate gene silencing (e.g. RNAi ortranslational repression).

As used herein, the term “bond strength” or “base pair strength” refersto the strength of the interaction between pairs of nucleotides (ornucleotide analogs) on opposing strands of an oligonucleotide duplex(e.g., an siRNA duplex), due primarily to H-bonding, Van der Waalsinteractions, and the like between said nucleotides (or nucleotideanalogs).

As used herein, the “5′ end”, as in the 5′ end of an antisense strand,refers to the 5′ terminal nucleotides, e.g., between one and about 5nucleotides at the 5′ terminus of the antisense strand. As used herein,the “3′ end”, as in the 3′ end of a sense strand, refers to the region,e.g., a region of between one and about 5 nucleotides, that iscomplementary to the nucleotides of the 5′ end of the complementaryantisense strand.

As used herein, the term “isolated RNA” (e.g., “isolated shRNA”,“isolated siRNA”, “isolated siRNA-like duplex”, “isolated miRNA”,“isolated gene silencing agent”, or “isolated RNAi agent”) refers to RNAmolecules which are substantially free of other cellular material, orculture medium when produced by recombinant techniques, or substantiallyfree of chemical precursors or other chemicals when chemicallysynthesized.

As used herein, the term “transgene” refers to any nucleic acidmolecule, which is inserted by artifice into a cell, and becomes part ofthe genome of the organism that develops from the cell. Such a transgenemay include a gene that is partly or entirely heterologous (i.e.,foreign) to the transgenic organism, or may represent a gene homologousto an endogenous gene of the organism. The term “transgene” also means anucleic acid molecule that includes one or more selected nucleic acidsequences, e.g., DNAs, that encode one or more engineered RNAprecursors, to be expressed in a transgenic organism, e.g., animal,which is partly or entirely heterologous, i.e., foreign, to thetransgenic animal, or homologous to an endogenous gene of the transgenicanimal, but which is designed to be inserted into the animal's genome ata location which differs from that of the natural gene. A transgeneincludes one or more promoters and any other DNA, such as introns,necessary for expression of the selected nucleic acid sequence, alloperably linked to the selected sequence, and may include an enhancersequence.

The term “in vitro” has its art recognized meaning, e.g., involvingpurified reagents or extracts, e.g., cell extracts. The term “in vivo”also has its art recognized meaning, e.g., involving living cells, e.g.,immortalized cells, primary cells, cell lines, and/or cells in anorganism.

A gene “involved” in a disorder includes a gene, the normal or aberrantexpression or function of which effects or causes a disease or disorderor at least one symptom of said disease or disorder.

Various methodologies of the instant invention include step thatinvolves comparing a value, level, feature, characteristic, property,etc. to a “suitable control”, referred to interchangeably herein as an“appropriate control”. A “suitable control” or “appropriate control” isany control or standard familiar to one of ordinary skill in the artuseful for comparison purposes. In one embodiment, a “suitable control”or “appropriate control” is a value, level, feature, characteristic,property, etc. determined prior to performing an RNAi methodology, asdescribed herein. For example, a transcription rate, mRNA level,translation rate, protein level, biological activity, cellularcharacteristic or property, genotype, phenotype, etc. can be determinedprior to introducing an RNAi agent of the invention into a cell ororganism. In another embodiment, a “suitable control” or “appropriatecontrol” is a value, level, feature, characteristic, property, etc.determined in a cell or organism, e.g., a control or normal cell ororganism, exhibiting, for example, normal traits. In yet anotherembodiment, a “suitable control” or “appropriate control” is apredefined value, level, feature, characteristic, property, etc.

Various aspects of the invention are described in further detail in thefollowing subsections.

I. RNA Silencing Agents

The present invention features RNA silencing agents (e.g. siRNAs orsiRNA-like duplexes), in particular, RNA silencing agents havingenhanced efficacy for gene silencing based on an asymmetry whichpromotes entry of a preferred strand into RISC.

1. Enhancement of RNA Silencing Agents

In certain aspects, the present invention features “small interferingRNA molecules” (“siRNA molecules” or “siRNA”), “miRNA” molecules,methods of making said siRNA and miRNA molecules, and methods (e.g.,research and/or therapeutic methods) for using said siRNA and miRNAmolecules. An RNA silencing agent (e.g. siRNA or miRNA molecule) of theinvention is a duplex consisting of a sense strand and complementaryantisense strand, the antisense strand having sufficient complementarityto a target mRNA to mediate RNAi or translational repression.Preferably, the strands are aligned such that there are at least 1, 2,or 3 bases at the end of the strands which do not align (i.e., for whichno complementary bases occur in the opposing strand) such that anoverhang of 1, 2 or 3 residues occurs at one or both ends of the duplexwhen strands are annealed. Preferably, the siRNA or miRNA molecule has alength from about 10-50 or more nucleotides, i.e., each strand comprises10-50 nucleotides (or nucleotide analogs). More preferably, the siRNA ormiRNA molecule has a length from about 15-45 or 15-30 nucleotides. Evenmore preferably, the siRNA or miRNA molecule has a length from about16-25 or 18-23 nucleotides.

In other aspects, the invention features siRNA-like duplex molecules, aswell as methods, for making and using such molecules in RNA silencing.siRNA-like duplexes (like siRNA duplexes) include a first or miRNAstrand and a second or miRNA* strand and are structurally similar tosiRNA duplexes. Modifications within the duplex are permitted.Modifications that do not significantly affect RNA-silencing activityare particularly tolerated. siRNA-like duplexes mediate translationalrepression or RNAi depending on the degree of complementarity withtarget mRNA, as described supra.

RNA silencing agents (e.g. siRNAs or miRNAs) featured in the inventionprovide enhanced specificity and efficacy for mediating RISC-mediatedcleavage or translational repression of a desired target gene. Inpreferred aspect, the base pair strength between the antisense strand 5′end (AS 5′) and the sense strand 3′ end (S 3′) of the siRNA, or miRNAsmolecule is less than the bond strength or base pair strength betweenthe antisense strand 3′ end (AS 3′) and the sense strand 5′ end (S 5′),such that the antisense strand preferentially guides cleavage ortranslational repression of a target mRNA.

In one embodiment, the bond strength or base-pair strength is less dueto fewer G:C base pairs between the 5′ end of the first or antisensestrand and the 3′ end of the second or sense strand than between the 3′end of the first or antisense strand and the 5′ end of the second orsense strand.

In another embodiment, the bond strength or base pair strength is lessdue to at least one mismatched base pair between the 5′ end of the firstor antisense strand and the 3′ end of the second or sense strand.Preferably, the mismatched base pair is selected from the groupconsisting of G:A, C:A, C:U, G:G, A:A, C:C and U:U. In a relatedembodiment, the bond strength or base pair strength is less due to atleast one wobble base pair, e.g., G:U, between the 5′ end of the firstor antisense strand and the 3′ end of the second or sense strand.

In yet another embodiment, the bond strength or base pair strength isless due to at least one base pair comprising a rare nucleotide, e.g,inosine (I). Preferably, the base pair is selected from the groupconsisting of an I:A, I:U and I:C.

In yet another embodiment, the bond strength or base pair strength isless due to at least one base pair comprising a modified nucleotide. Inpreferred embodiments, the modified nucleotide is selected from thegroup consisting of 2-amino-G, 2-amino-A, 2,6-diamino-G, and2,6-diamino-A.

2. Complementarity of RNA Silencing Agents with Target mRNA

The siRNA molecules of the invention further have a sequence that is“sufficiently complementary” to a target mRNA sequence to directtarget-specific RNA interference (RNAi), as defined herein, i.e., thesiRNA has a sequence sufficient to trigger the destruction of the targetmRNA by the RNAi machinery or process.

In general, siRNA containing nucleotide sequences sufficiently identicalto a portion of the target gene to effect RISC-mediated cleavage of thetarget gene are preferred. 100% sequence identity between the siRNA andthe target gene is not required to practice the present invention. Theinvention has the advantage of being able to tolerate certain sequencevariations to enhance efficiency and specificity of RNAi. Moreover,siRNA sequences with insertions, deletions, and single point mutationsrelative to the target sequence can also be effective for inhibition.Alternatively, siRNA sequences with nucleotide analog substitutions orinsertions can be effective for inhibition.

siRNA-like molecules of the invention have a sequence (i.e., have astrand having a sequence) that is “sufficiently complementary” to atarget mRNA sequence to direct gene silencing either by RNAi ortranslational repression. As the degree of sequence identity between amiRNA sequence and the corresponding target gene sequence is decreased,the tendency to mediate post-transcriptional gene silencing bytranslational repression rather than RNAi is increased. Therefore, in analternative embodiment, where post-transcriptional gene silencing bytranslational repression of the target gene is desired, the miRNAsequence has partial complementarity with the target gene sequence. Incertain embodiments, the miRNA sequence has partial complementarity withone or more short sequences (complementarity sites) dispersed within thetarget mRNA (e.g. within the 3′-UTR of the target mRNA) (Hutvàgner andZamore, Science, 2002; Zeng et al., Mol. Cell, 2002; Zeng et al., RNA,2003; Doench et al., Genes & Dev., 2003). Since the mechanism oftranslational repression is cooperative, multiple complementarity sites(e.g., 2, 3, 4, 5, or 6) may be targeted in certain embodiments.

Sequence identity may determined by sequence comparison and alignmentalgorithms known in the art. To determine the percent identity of twonucleic acid sequences (or of two amino acid sequences), the sequencesare aligned for optimal comparison purposes (e.g., gaps can beintroduced in the first sequence or second sequence for optimalalignment). The nucleotides (or amino acid residues) at correspondingnucleotide (or amino acid) positions are then compared. When a positionin the first sequence is occupied by the same residue as thecorresponding position in the second sequence, then the molecules areidentical at that position. The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences (i.e., % homology=# of identical positions/total # ofpositions×100), optionally penalizing the score for the number of gapsintroduced and/or length of gaps introduced.

The comparison of sequences and determination of percent identitybetween two sequences can be accomplished using a mathematicalalgorithm. In one embodiment, the alignment generated over a certainportion of the sequence aligned having sufficient identity but not overportions having low degree of identity (i.e., a local alignment). Apreferred, non-limiting example of a local alignment algorithm utilizedfor the comparison of sequences is the algorithm of Karlin and Altschul(1990) Proc. Natl. Acad. Sci. USA 87:2264-68, modified as in Karlin andAltschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-77. Such an algorithmis incorporated into the BLAST programs (version 2.0) of Altschul, etal. (1990) J. Mol. Biol. 215:403-10.

In another embodiment, the alignment is optimized by introducingappropriate gaps and percent identity is determined over the length ofthe aligned sequences (i.e., a gapped alignment). To obtain gappedalignments for comparison purposes, Gapped BLAST can be utilized asdescribed in Altschul et al., (1997) Nucleic Acids Res.25(17):3389-3402. In another embodiment, the alignment is optimized byintroducing appropriate gaps and percent identity is determined over theentire length of the sequences aligned (i.e., a global alignment). Apreferred, non-limiting example of a mathematical algorithm utilized forthe global comparison of sequences is the algorithm of Myers and Miller,CABIOS (1989). Such an algorithm is incorporated into the ALIGN program(version 2.0) which is part of the GCG sequence alignment softwarepackage. When utilizing the ALIGN program for comparing amino acidsequences, a PAM120 weight residue table, a gap length penalty of 12,and a gap penalty of 4 can be used.

Greater than 80% sequence identity, e.g., 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% oreven 100% sequence identity, between the siRNA antisense strand and theportion of the target gene is preferred. Conversely, miRNA sequenceswith less than 80% identity and the portion of the target gene (i.e. thesite of complementarity) are preferred in order to mediate silencing bytranslational repression. Alternatively, the siRNA may be definedfunctionally as a nucleotide sequence (or oligonucleotide sequence) thatis capable of hybridizing with a portion of the target gene transcript(e.g., 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50° C. or 70° C.hybridization for 12-16 hours; followed by washing). Additionalpreferred hybridization conditions include hybridization at 70° C. in1×SSC or 50° C. in 1×SSC, 50% formamide followed by washing at 70° C. in0.3×SSC or hybridization at 70° C. in 4×SSC or 50° C. in 4×SSC, 50%formamide followed by washing at 67° C. in 1×SSC. The hybridizationtemperature for hybrids anticipated to be less than 50 base pairs inlength should be 5-10° C. less than the melting temperature (Tm) of thehybrid, where Tm is determined according to the following equations. Forhybrids less than 18 base pairs in length, Tm(° C.)=2(# of A+Tbases)+4(# of G+C bases). For hybrids between 18 and 49 base pairs inlength, Tm(° C.)=81.5+16.6(log 10[Na+])+0.41(% G+C)−(600/N), where N isthe number of bases in the hybrid, and [Na+] is the concentration ofsodium ions in the hybridization buffer ([Na+] for 1×SSC=0.165 M).Additional examples of stringency conditions for polynucleotidehybridization are provided in Sambrook, J., E. F. Fritsch, and T.Maniatis, 1989, Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., chapters 9 and 11,and Current Protocols in Molecular Biology, 1995, F. M. Ausubel et al.,eds., John Wiley & Sons, Inc., sections 2.10 and 6.3-6.4, incorporatedherein by reference. The length of the identical nucleotide sequencesmay be at least about 10, 12, 15, 17, 20, 22, 25, 27, 30, 32, 35, 37,40, 42, 45, 47 or 50 bases.

The capacity of a siRNA-like duplex to mediate RNAi or translationalrepression may be predicted by the distribution of non-identicalnucleotides between the target gene sequence and the nucleotide sequenceof the silencing agent at the site of complementarity. In oneembodiment, where gene silencing by translational repression is desired,at least one non-identical nucleotide is present in the central portionof the complementarity site so that duplex formed by the miRNA guidestrand and the target mRNA contains a central “bulge” (Doench J G etal., Genes & Dev., 2003). In another embodiment 2, 3, 4, 5, or 6contiguous or non-contiguous non-identical nucleotides are introduced.The non-identical nucleotide may be selected such that it forms a wobblebase pair (e.g., G:U) or a mismatched base pair (G:A, C:A, C:U, G:G,A:A, C:C, U:U). In a further preferred embodiment, the “bulge” iscentered at nucleotide positions 12 and 13 from the 5′ end of the miRNAmolecule.

3. Modification of RNA Silencing Agents

The RNA silencing molecules of the present invention can be modified toimprove stability in serum or in growth medium for cell cultures. Inorder to enhance the stability, the 3′-residues may be stabilizedagainst degradation, e.g., they may be selected such that they consistof purine nucleotides, particularly adenosine or guanosine nucleotides.Alternatively, substitution of pyrimidine nucleotides by modifiedanalogues, e.g., substitution of uridine by 2′-deoxythymidine istolerated and does not affect the efficiency of RNA interference.

In a preferred aspect, the invention features RNA silencing agents (e.g.small interfering RNAs (siRNAs) or siRNA-like molecules) that includefirst and second strands wherein the second strand and/or first strandis modified by the substitution of internal nucleotides with modifiednucleotides, such that in vivo stability is enhanced as compared to acorresponding unmodified agent (e.g. siRNA or siRNA-like duplex). Asdefined herein, an “internal” nucleotide is one occurring at anyposition other than the 5′ end or 3′ end of nucleic acid molecule,polynucleotide or oligonucleotide. An internal nucleotide can be withina single-stranded molecule or within a strand of a duplex ordouble-stranded molecule. In one embodiment, the sense strand and/orantisense strand is modified by the substitution of at least oneinternal nucleotide. In another embodiment, the sense strand and/orantisense strand is modified by the substitution of at least 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25 or more internal nucleotides. In another embodiment, the sense strandand/or antisense strand is modified by the substitution of at least 5%,10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95% or more of the internal nucleotides. In yet anotherembodiment, the sense strand and/or antisense strand is modified by thesubstitution of all of the internal nucleotides.

In a preferred embodiment of the present invention the RNA molecule maycontain at least one modified nucleotide analogue. The nucleotideanalogues may be located at positions where the target-specificsilencing activity, e.g., the RNAi mediating activity or translationalrepression activity is not substantially effected, e.g., in a region atthe 5′-end and/or the 3′-end of the RNA molecule. Particularly, the endsmay be stabilized by incorporating modified nucleotide analogues.

Preferred nucleotide analogues include sugar- and/or backbone-modifiedribonucleotides (i.e., include modifications to the phosphate-sugarbackbone). For example, the phosphodiester linkages of natural RNA maybe modified to include at least one of a nitrogen or sulfur heteroatom.In preferred backbone-modified ribonucleotides the phosphoester groupconnecting to adjacent ribonucleotides is replaced by a modified group,e.g., of phosphothioate group. In preferred sugar-modifiedribonucleotides, the 2′ OH-group is replaced by a group selected from H,OR, R, halo, SH, SR, NH₂, NHR, NR₂ or ON, wherein R is C₁-C₆ alkyl,alkenyl or alkynyl and halo is F, Cl, Br or I.

Also preferred are nucleobase-modified ribonucleotides, i.e.,ribonucleotides, containing at least one non-naturally occurringnucleobase instead of a naturally occurring nucleobase. Bases may bemodified to block the activity of adenosine deaminase. Exemplarymodified nucleobases include, but are not limited to, uridine and/orcytidine modified at the 5-position, e.g., 5-(2-amino)propyl uridine,5-bromo uridine; adenosine and/or guanosines modified at the 8 position,e.g., 8-bromo guanosine; deaza nucleotides, e.g., 7-deaza-adenosine; O-and N-alkylated nucleotides, e.g., N6-methyl adenosine are suitable. Itshould be noted that the above modifications may be combined.

4. Production of RNA Silencing Agents

RNA may be produced enzymatically or by partial/total organic synthesis,any modified ribonucleotide can be introduced by in vitro enzymatic ororganic synthesis. In one embodiment, a silencing agent (e.g. a RNAiagent or translational repression agent) is prepared chemically. Methodsof synthesizing RNA molecules are known in the art, in particular, thechemical synthesis methods as described in Verma and Eckstein (1998)Annul Rev. Biochem. 67:99-134.

In one embodiment, a RNA silencing agent (e.g. siRNA or siRNA-likeduplex) is prepared enzymatically. For example, a short duplex RNA (e.g.siRNA or siRNA-like duplex) can be prepared by enzymatic processing of along ds RNA having sufficient complementarity to the desired targetmRNA. Processing of long ds RNA can be accomplished in vitro, forexample, using appropriate cellular lysates and duplex RNA (e.g. siRNAor siRNA-like duplex) can be subsequently purified by gelelectrophoresis or gel filtration. Duplex RNA (e.g. siRNA or siRNA-likeduplex) can then be denatured according to art-recognized methodologies.In an exemplary embodiment, RNA can be purified from a mixture byextraction with a solvent or resin, precipitation, electrophoresis,chromatography, or a combination thereof. Alternatively, the RNA may beused with no or a minimum of purification to avoid losses due to sampleprocessing. Alternatively, the RNA can also be prepared by enzymatictranscription from synthetic DNA templates or from DNA plasmids isolatedfrom recombinant bacteria. Typically, phage RNA polymerases are usedsuch as T7, T3 or SP6 RNA polymerase (Milligan and Uhlenbeck (1989)Methods Enzymol. 180:51-62). The RNA may be dried for storage ordissolved in an aqueous solution. The solution may contain buffers orsalts to inhibit annealing, and/or promote stabilization of the singlestrands.

RNA silencing agents of the invention can also be prepared in vivo byenzymatic processing of a long dsRNA molecule (>30 b.p.) which hassufficient complementarity to the desired target mRNA. Preferably, invivo processing of the long dsRNA molecule occurs in a non-mammaliancell or a mammalian cell which is deficient in the interferon-mediatedinflammatory response to dsRNA. In one embodiment, the cell capable ofdsRNA enzymatic processing may be present within an organism such thatdsRNA processing can be induced in vivo to trigger gene silencing of atarget gene within the organism. Alternatively, the cell (i.e. a hostcell) containing endogenous machinery for dsRNA processing (e.g. DICER)or transformed with heterologous genes to enable dsRNA processing) becultured and induced to process dsRNA in vitro. RNA silencing agents maythen be purified from the host cell following dsRNA processing foradministration to an organism containing the target gene to be silenced.

In another embodiment, RNA silencing agents (e.g. siRNAs or siRNA-likeduplexes) are synthesized directly either in vivo, in situ, or in vitro.An endogenous RNA polymerase in the cell may mediate transcription ofthe RNA silencing agent in vivo or in situ, or a cloned RNA polymerasecan be used for transcription of the RNA silencing agent in vivo or invitro. For transcription from a transgene in vivo or an expressionconstruct, a regulatory region (e.g., promoter, enhancer, silencer,splice donor and acceptor, polyadenylation) may be used to transcribethe RNA silencing agent (e.g. siRNA or siRNA-like duplexes). Inhibitionmay be targeted by specific transcription in an organ, tissue, or celltype; stimulation of an environmental condition (e.g., infection,stress, temperature, chemical inducers); and/or engineeringtranscription at a developmental stage or age. A transgenic organismthat expresses a RNA silencing agent (e.g. siRNA or siRNA-like duplexes)from a recombinant construct may be produced by introducing theconstruct into a zygote, an embryonic stem cell, or another multipotentcell derived from the appropriate organism.

5. Targeted mRNAs of RNA Silencing Agents

In one embodiment, the target mRNA of the invention specifies the aminoacid sequence of a cellular protein (e.g., a nuclear, cytoplasmic,transmembrane, or membrane-associated protein). In another embodiment,the target mRNA of the invention specifies the amino acid sequence of anextracellular protein (e.g., an extracellular matrix protein or secretedprotein). As used herein, the phrase “specifies the amino acid sequence”of a protein means that the mRNA sequence is translated into the aminoacid sequence according to the rules of the genetic code. The followingclasses of proteins are listed for illustrative purposes: developmentalproteins (e.g., adhesion molecules, cyclin kinase inhibitors, Wnt familymembers, Pax family members, Winged helix family members, Hox familymembers, cytokines/lymphokines and their receptors,growth/differentiation factors and their receptors, neurotransmittersand their receptors); oncogene-encoded proteins (e.g., ABLI, BCLI, BCL2,BCL6, CBFA2, CBL, CSFIR, ERBA, ERBB, EBRB2, ETSI, ETSI, ETV6, FGR, FOS,FYN, HCR, HRAS, JUN, KRAS, LCK, LYN, MDM2, MLL, MYB, MYC, MYCLI, MYCN,NRAS, PIM I, PML, RET, SRC, TALI, TCL3, and YES); tumor suppressorproteins (e.g., APC, BRCA1, BRCA2, MADH4, MCC, NF I, NF2, RB I, TP53,and WTI); and enzymes (e.g., ACC synthases and oxidases, ACP desaturasesand hydroxylases, ADP-glucose pyrophorylases, ATPases, alcoholdehydrogenases, amylases, amyloglucosidases, catalases, cellulases,chalcone synthases, chitinases, cyclooxygenases, decarboxylases,dextriinases, DNA and RNA polymerases, galactosidases, glucanases,glucose oxidases, granule-bound starch synthases, GTPases, helicases,hernicellulases, integrases, inulinases, invertases, isomerases,kinases, lactases, lipases, lipoxygenases, lysozymes, nopalinesynthases, octopine synthases, pectinesterases, peroxidases,phosphatases, phospholipases, phosphorylases, phytases, plant growthregulator synthases, polygalacturonases, proteinases and peptidases,pullanases, recombinases, reverse transcriptases, RUBISCOs,topoisomerases, and xylanases).

In a preferred aspect of the invention, the target mRNA molecule of theinvention specifies the amino acid sequence of a protein associated witha pathological condition. For example, the protein may be apathogen-associated protein (e.g., a viral protein involved inimmunosuppression of the host, replication of the pathogen, transmissionof the pathogen, or maintenance of the infection), or a host proteinwhich facilitates entry of the pathogen into the host, drug metabolismby the pathogen or host, replication or integration of the pathogen'sgenome, establishment or spread of infection in the host, or assembly ofthe next generation of pathogen. Alternatively, the protein may be atumor-associated protein or an autoimmune disease-associated protein.

In one embodiment, the target mRNA molecule of the invention specifiesthe amino acid sequence of an endogenous protein (i.e., a proteinpresent in the genome of a cell or organism). In another embodiment, thetarget mRNA molecule of the invention specified the amino acid sequenceof a heterologous protein expressed in a recombinant cell or agenetically altered organism. In another embodiment, the target mRNAmolecule of the invention specified the amino acid sequence of a proteinencoded by a transgene (i.e., a gene construct inserted at an ectopicsite in the genome of the cell). In yet another embodiment, the targetmRNA molecule of the invention specifies the amino acid sequence of aprotein encoded by a pathogen genome which is capable of infecting acell or an organism from which the cell is derived.

By inhibiting the expression of such proteins, valuable informationregarding the function of said proteins and therapeutic benefits whichmay be obtained from said inhibition may be obtained.

II. Short Hairpin RNAs (shRNAs)

In certain featured embodiments, the instant invention provides shRNAshaving enhanced specificity or efficacy in mediating gene silencing(e.g. RNAi or translational repression). In contrast to short RNAsilencing duplexes (e.g. siRNA or siRNA-like duplexes), short hairpinRNAs (shRNAs) mimic the natural precursors of miRNAs and enter at thetop of the gene silencing pathway. For this reason, shRNAs are believedto mediate gene silencing more efficiently by being fed through theentire natural gene silencing pathway.

A preferred shRNA of the invention is one that has been redesigned forincreased specificity or enhancement relative to a previous shRNA. Thenew shRNA differs from a previous shRNA in that a silencing duplexproduced from the new shRNA has less base pair strength between the 5′end of the antisense strand or first strand and the 3′ end of the sensestrand or second strand than the base pair strength between the 3′ endof the antisense strand or first strand and the 5′ end of the sensestrand or second strand.

1. Engineered RNA Precursors that Generate RNA Silencing Agents

Naturally-occurring miRNA precursors (pre-miRNA) have a single strandthat forms a duplex stem including two portions that are generallycomplementary, and a loop, that connects the two portions of the stem.In typical pre-miRNAs, the stem includes one or more bulges, e.g., extranucleotides that create a single nucleotide “loop” in one portion of thestem, and/or one or more unpaired nucleotides that create a gap in thehybridization of the two portions of the stem to each other. Shorthairpin RNAs, or engineered RNA precursors, of the invention areartificial constructs based on these naturally occurring pre-miRNAs, butwhich are engineered to deliver desired RNA silencing agents (e.g.,siRNAs or siRNA-like duplexes).

In shRNAs, or engineered precursor RNAs, of the instant invention, oneportion of the duplex stem is a nucleic acid sequence that iscomplementary (or anti-sense) to the target mRNA. Thus, engineered RNAprecursors include a duplex stem with two portions and a loop connectingthe two stem portions. The two stem portions are about 18 or 19 to about25, 30, 35, 37, 38, 39, or 40 or more nucleotides in length. When usedin mammalian cells, the length of the stem portions should be less thanabout 30 nucleotides to avoid provoking non-specific responses like theinterferon pathway. In non-mammalian cells, the stem can be longer than30 nucleotides. In fact, the stem can include much larger sectionscomplementary to the target mRNA (up to, and including the entire mRNA).The two portions of the duplex stem must be sufficiently complementaryto hybridize to form the duplex stem. Thus, the two portions can be, butneed not be, fully or perfectly complementary. In addition, the two stemportions can be the same length, or one portion can include an overhangof 1, 2, 3, or 4 nucleotides. The overhanging nucleotides can include,for example, uracils (Us), e.g., all Us. The loop in the shRNAs orengineered RNA precursors may differ from natural pre-miRNA sequences bymodifying the loop sequence to increase or decrease the number of pairednucleotides, or replacing all or part of the loop sequence with atetraloop or other loop sequences. Thus, the loop in the shRNAs orengineered RNA precursors can be 2, 3, 4, 5, 6, 7, 8, 9, or more, e.g.,15 or 20, or more nucleotides in length.

In certain embodiments, shRNAs of the invention include the sequences ofa desired RNA silencing agent (e.g. siRNA or siRNA-like duplex). Thedesired RNA silencing duplex (e.g. siRNA or siRNA-like duplex), and thusboth of the two stem portions in the engineered RNA precursor, areselected by methods known in the art. These include, but are not limitedto, selecting an 18, 19, 20, 21 nucleotide, or longer, sequence from thetarget gene mRNA sequence from a region 100 to 200 or 300 nucleotides onthe 3′ side of the start of translation. In general, the sequence can beselected from any portion of the mRNA from the target gene, such as the5′ UTR (untranslated region), coding sequence, or 3′ UTR. This sequencecan optionally follow immediately after a region of the target genecontaining two adjacent AA nucleotides. The last two nucleotides of the21 or so nucleotide sequence can be selected to be UU (so that theanti-sense strand of the siRNA begins with UU). This 21 or so nucleotidesequence is used to create one portion of a duplex stem in theengineered RNA precursor. This sequence can replace a stem portion of awild-type pre-stRNA sequence, e.g., enzymatically, or is included in acomplete sequence that is synthesized. For example, one can synthesizeDNA oligonucleotides that encode the entire stem-loop engineered RNAprecursor, or that encode just the portion to be inserted into theduplex stem of the precursor, and using restriction enzymes to build theengineered RNA precursor construct, e.g., from a wild-type pre-stRNA.

Engineered RNA precursors include in the duplex stem the 21-22 or sonucleotide sequences of the siRNA or siRNA-like duplex desired to beproduced in vivo. Thus, the stem portion of the engineered RNA precursorincludes at least 18 or 19 nucleotide pairs corresponding to thesequence of an exonic portion of the gene whose expression is to bereduced or inhibited. The two 3′ nucleotides flanking this region of thestem are chosen so as to maximize the production of the siRNA from theengineered RNA precursor, and to maximize the efficacy of the resultingsiRNA in targeting the corresponding mRNA for translational repressionor destruction by RNAi in vivo and in vitro.

In certain embodiments, shRNAs of the invention include miRNA sequences,optionally end-modified miRNA sequences, to enhance entry into RISC. ThemiRNA sequence can be similar or identical to that of any naturallyoccurring miRNA (see e.g. The miRNA Registry: Griffiths-Jones S, Nuc.Acids Res., 2004). Over one thousand natural miRNAs have been identifiedto date and together they are thought to comprise ˜1% of all predictedgenes in the genome. Many natural miRNAs are clustered together in theintrons of pre-mRNAs and can be identified in silico usinghomology-based searches (Pasquinelli et al., 2000; Lagos-Quintana etal., 2001; Lau et al., 2001; Lee and Ambros, 2001) or computeralgorithms (e.g. MiRScan, MiRSeeker) that predict the capability of acandidate miRNA gene to form the stem loop structure of a pri-mRNA (Gradet al., Mol. Cell., 2003; Lim et al., Genes Dev., 2003; Lim et al.,Science, 2003; Lai E C et al., Genome Bio., 2003). An online registryprovides a searchable database of all published miRNA sequences (ThemiRNA Registry at the Sanger Institute website; Griffiths-Jones S, Nuc.Acids Res., 2004). Exemplary, natural miRNAs include lin-4, let-7,miR-10, mirR-15, miR-16, miR-168, miR-175, miR-196 and their homologs,as well as other natural miRNAs from humans and certain model organismsincluding Drosophila melanogaster, Caenorhabditis elegans, zebrafish,Arabidopsis thalania, mouse, and rat as described in International PCTPublication No. WO 03/029459.

Naturally-occurring miRNAs are expressed by endogenous genes in vivo andare processed from a hairpin or stem-loop precursor (pre-miRNA orpri-miRNAs) by Dicer or other RNAses (Lagos-Quintana et al., Science,2001; Lau et al., Science, 2001; Lee and Ambros, Science, 2001;Lagos-Quintana et al., Curr. Biol., 2002; Mourelatos et al., Genes Dev.,2002; Reinhart et al., Science, 2002; Ambros et al., Curr. Biol., 2003;Brennecke et al., 2003; Lagos-Quintana et al., RNA, 2003; Lim et al.,Genes Dev., 2003; Lim et al., Science, 2003). miRNAs can existtransiently in vivo as a double-stranded duplex but only one strand istaken up by the RISC complex to direct gene silencing. Certain miRNAs,e.g. plant miRNAs, have perfect or near-perfect complementarity to theirtarget mRNAs and, hence, direct cleavage of the target mRNAs. OthermiRNAs have less than perfect complementarity to their target mRNAs and,hence, direct translational repression of the target mRNAs. The degreeof complementarity between an miRNA and its target mRNA is believed todetermine its mechanism of action. For example, perfect or near-perfectcomplementarity between a miRNA and its target mRNA is predictive of acleavage mechanism (Yekta et al., Science, 2004), whereas less thanperfect complementarity is predictive of a translational repressionmechanism.

In particular embodiments, the miRNA sequence is that of anaturally-occurring miRNA sequence, the aberrant expression or activityof which is correlated with a miRNA disorder.

Another defining feature of these engineered RNA precursors is that as aconsequence of their length, sequence, and/or structure, they do notinduce sequence non-specific responses, such as induction of theinterferon response or apoptosis, or that they induce a lower level ofsuch sequence non-specific responses than long, double-stranded RNA(>150 bp) that has been used to induce post-transcriptional genesilencing (e.g. RNAi). For example, the interferon response is triggeredby dsRNA longer than 30 base pairs.

2. Transgenes Encoding Engineered RNA Precursors

The new engineered RNA precursors can be synthesized by standard methodsknown in the art, e.g., by use of an automated DNA synthesizer (such asare commercially available from Biosearch, Applied Biosystems, etc.).These synthetic, engineered RNA precursors can be used directly asdescribed below or cloned into expression vectors by methods known inthe field. The engineered RNA precursors should be delivered to cells invitro or in vivo in which it is desired to target a specific mRNA forsilencing (e.g. destruction or repression). A number of methods havebeen developed for delivering DNA or RNA to cells. For example, for invivo delivery, molecules can be injected directly into a tissue site oradministered systemically. In vitro delivery includes methods known inthe art such as electroporation and lipofection.

To achieve intracellular concentrations of the nucleic acid moleculesufficient to suppress expression of endogenous mRNAs, one can use, forexample, a recombinant DNA construct in which the oligonucleotide isplaced under the control of a strong Pol III (e.g., U6 or PolIII H1-RNApromoter) or Pol II promoter. The use of such a construct to transfecttarget cells in vitro or in vivo will result in the transcription ofsufficient amounts of the engineered RNA precursor to lead to theproduction of a RNA duplex (e.g. siRNA or siRNA-like duplex) that cantarget a corresponding mRNA sequence for cleavage (i.e. RNAi) ortranslational repression to decrease the expression of the gene encodingthat mRNA. For example, a vector can be introduced in vivo such that itis taken up by a cell and directs the transcription of an engineered RNAprecursor. Such a vector can remain episomal or become chromosomallyintegrated, as long as it can be transcribed to produce the desiredstRNA precursor.

Such vectors can be constructed by recombinant DNA technology methodsknown in the art. Vectors can be plasmid, viral, or other vectors knownin the art such as those described herein, used for replication andexpression in mammalian cells or other targeted cell types. The nucleicacid sequences encoding the engineered RNA precursors can be preparedusing known techniques. For example, two synthetic DNA oligonucleotidescan be synthesized to create a novel gene encoding the entire engineeredRNA precursor. The DNA oligonucleotides, which will pair, leavingappropriate ‘sticky ends’ for cloning, can be inserted into arestriction site in a plasmid that contains a promoter sequence (e.g., aPol II or a Pol III promoter) and appropriate terminator sequences 3′ tothe engineered RNA precursor sequences (e.g., a cleavage andpolyadenylation signal sequence from SV40 or a Pol III terminatorsequence).

The invention also encompasses genetically engineered host cells thatcontain any of the foregoing expression vectors and thereby express thenucleic acid molecules of the invention in the host cell. The host cellscan be cultured using known techniques and methods (see, e.g., Cultureof Animal Cells (R. I. Freshney, Alan R. Liss, Inc. 1987); MolecularCloning, Sambrook et al. (Cold Spring Harbor Laboratory Press, 1989)).

Successful introduction of the vectors of the invention into host cellscan be monitored using various known methods. For example, transienttransfection can be signaled with a reporter, such as a fluorescentmarker, such as Green Fluorescent Protein (GFP). Stable transfection canbe indicated using markers that provide the transfected cell withresistance to specific environmental factors (e.g., antibiotics anddrugs), such as hygromycin B resistance, e.g., in insect cells and inmammalian cells.

3. Regulatory Sequences

The expression of the engineered RNA precursors is driven by regulatorysequences, and the vectors of the invention can include any regulatorysequences known in the art to act in mammalian cells, e.g., human ormurine cells; in insect cells; in plant cells; or other cells. The termregulatory sequence includes promoters, enhancers, and other expressioncontrol elements. It will be appreciated that the appropriate regulatorysequence depends on such factors as the future use of the cell ortransgenic animal into which a sequence encoding an engineered RNAprecursor is being introduced, and the level of expression of thedesired RNA precursor. A person skilled in the art would be able tochoose the appropriate regulatory sequence. For example, the transgenicanimals described herein can be used to determine the role of a testpolypeptide or the engineered RNA precursors in a particular cell type,e.g., a hematopoietic cell. In this case, a regulatory sequence thatdrives expression of the transgene ubiquitously, or ahematopoietic-specific regulatory sequence that expresses the transgeneonly in hematopoietic cells, can be used. Expression of the engineeredRNA precursors in a hematopoietic cell means that the cell is nowsusceptible to specific, targeted silencing (e.g. RNAi) of a particulargene. Examples of various regulatory sequences are described below.

The regulatory sequences can be inducible or constitutive. Suitableconstitutive regulatory sequences include the regulatory sequence of ahousekeeping gene such as the α-actin regulatory sequence, or may be ofviral origin such as regulatory sequences derived from mouse mammarytumor virus (MMTV) or cytomegalovirus (CMV).

Alternatively, the regulatory sequence can direct transgene expressionin specific organs or cell types (see, e.g., Lasko et al., 1992, Proc.Natl. Acad. Sci. USA 89:6232). Several tissue-specific regulatorysequences are known in the art including the albumin regulatory sequencefor liver (Pinkert et al., 1987, Genes Dev. 1:268-276); the endothelinregulatory sequence for endothelial cells (Lee, 1990, J. Biol. Chem.265:10446-50); the keratin regulatory sequence for epidermis; the myosinlight chain-2 regulatory sequence for heart (Lee et al., 1992, J. Biol.Chem. 267:15875-85), and the insulin regulatory sequence for pancreas(Bucchini et al., 1986, Proc. Natl. Acad. Sci. USA 83:2511-2515), or thevav regulatory sequence for hematopoietic cells (Oligvy et al., 1999,Proc. Natl. Acad. Sci. USA 96:14943-14948). Another suitable regulatorysequence, which directs constitutive expression of transgenes in cellsof hematopoietic origin, is the murine MHC class I regulatory sequence(Morello et al., 1986, EMBO J. 5:1877-1882). Since NMC expression isinduced by cytokines, expression of a test gene operably linked to thisregulatory sequence can be upregulated in the presence of cytokines.

In addition, expression of the transgene can be precisely regulated, forexample, by using an inducible regulatory sequence and expressionsystems such as a regulatory sequence that is sensitive to certainphysiological regulators, e.g., circulating glucose levels, or hormones(Docherty et al., 1994, FASEB J. 8:20-24). Such inducible expressionsystems, suitable for the control of transgene expression in cells or inmammals such as mice, include regulation by ecdysone, by estrogen,progesterone, tetracycline, chemical inducers of dimerization, andisopropyl-beta-D1-thiogalactopyranoside (IPTG) (collectively referred toas “the regulatory molecule”). Each of these expression systems is welldescribed in the literature and permits expression of the transgenethroughout the animal in a manner controlled by the presence or absenceof the regulatory molecule. For a review of inducible expressionsystems, see, e.g., Mills, 2001, Genes Devel. 15:1461-1467, andreferences cited therein.

The regulatory elements referred to above include, but are not limitedto, the cytomegalovirus hCMV immediate early gene, the early or latepromoters of SV40 adenovirus (Bernoist et al., Nature, 290:304, 1981),the tet system, the lac system, the system, the TAC system, the TRCsystem, the major operator and promoter regions of phage A, the controlregions of fd coat protein, the promoter for 3-phosphoglycerate kinase,the promoters of acid phosphatase, and the promoters of the yeastα-mating factors. Additional promoters include the promoter contained inthe 3′ long terminal repeat of Rous sarcoma virus (Yamamoto et al., Cell22:787-797, 1988); the herpes thymidine kinase promoter (Wagner et al.,Proc. Natl. Acad. Sci. USA 78:1441, 1981); or the regulatory sequencesof the metallothionein gene (Brinster et al., Nature 296:39, 1988).

4. Assay for Testing Engineered RNA Precursors

Drosophila embryo lysates can be used to determine if an engineered RNAprecursor was, in fact, the direct precursor of a mature stRNA or siRNA.This lysate assay is described in Tuschl et al., 1999, supra, Zamore etal., 2000, supra, and Hutvdgner et al. 2001, supra. These lysatesrecapitulate RNAi in vitro, thus permitting investigation into whetherthe proposed precursor RNA was cleaved into a mature stRNA or siRNA byan RNAi-like mechanism. Briefly, the precursor RNA is incubated withDrosophila embryo lysate for various times, then assayed for theproduction of the mature siRNA or stRNA by primer extension or Northernhybridization. As in the in vivo setting, mature RNA accumulates in thecell-free reaction. Thus, an RNA corresponding to the proposed precursorcan be shown to be converted into a mature stRNA or siRNA duplex in theDrosophila embryo lysate.

Furthermore, an engineered RNA precursor can be functionally tested inthe Drosophila embryo lysates. In this case, the engineered RNAprecursor is incubated in the lysate in the presence of a 5′radiolabeled target mRNA in a standard in vitro RNAi reaction forvarious lengths of time. The target mRNA can be 5′ radiolabeled usingguanylyl transferase (as described in Tuschl et al., 1999, supra andreferences therein) or other suitable methods. The products of the invitro reaction are then isolated and analyzed on a denaturing acrylamideor agarose gel to determine if the target mRNA has been cleaved inresponse to the presence of the engineered RNA precursor in thereaction. The extent and position of such cleavage of the mRNA targetwill indicate if the engineering of the precursor created a pre-siRNAcapable of mediating sequence-specific RNAi.

IV. Methods of Introducing RNAs, Vectors, and Host Cells

Physical methods of introducing nucleic acids include injection of asolution containing the RNA, bombardment by particles covered by theRNA, soaking the cell or organism in a solution of the RNA, orelectroporation of cell membranes in the presence of the RNA. A viralconstruct packaged into a viral particle would accomplish both efficientintroduction of an expression construct into the cell and transcriptionof RNA encoded by the expression construct. Other methods known in theart for introducing nucleic acids to cells may be used, such aslipid-mediated carrier transport, chemical-mediated transport, such ascalcium phosphate, and the like. Thus the RNA may be introduced alongwith components that perform one or more of the following activities:enhance RNA uptake by the cell, inhibit annealing of single strands,stabilize the single strands, or other-wise increase inhibition of thetarget gene.

RNA may be directly introduced into the cell (i.e., intracellularly); orintroduced extracellularly into a cavity, interstitial space, into thecirculation of an organism, introduced orally, or may be introduced bybathing a cell or organism in a solution containing the RNA. Vascular orextravascular circulation, the blood or lymph system, and thecerebrospinal fluid are sites where the RNA may be introduced.

The cell with the target gene may be derived from or contained in anyorganism. The organism may a plant, animal, protozoan, bacterium, virus,or fungus. The plant may be a monocot, dicot or gymnosperm; the animalmay be a vertebrate or invertebrate. Preferred microbes are those usedin agriculture or by industry, and those that are pathogenic for plantsor animals. Fungi include organisms in both the mold and yeastmorphologies. Plants include arabidopsis; field crops (e.g., alfalfa,barley, bean, corn, cotton, flax, pea, rape, nice, rye, safflower,sorghum, soybean, sunflower, tobacco, and wheat); vegetable crops (e.g.,asparagus, beet, broccoli, cabbage, carrot, cauliflower, celery,cucumber, eggplant, lettuce, onion, pepper, potato, pumpkin, radish,spinach, squash, taro, tomato, and zucchini); fruit and nut crops (e.g.,almond, apple, apricot, banana, black-berry, blueberry, cacao, cherry,coconut, cranberry, date, faJoa, filbert, grape, grapefruit, guava,kiwi, lemon, lime, mango, melon, nectarine, orange, papaya, passionfruit, peach, peanut, pear, pineapple, pistachio, plum, raspberry,strawberry, tangerine, walnut, and watermelon); and ornamentals (e.g.,alder, ash, aspen, azalea, birch, boxwood, camellia, carnation,chrysanthemum, elm, fir, ivy, jasmine, juniper, oak, palm, poplar, pine,redwood, rhododendron, rose, and rubber). Examples of vertebrate animalsinclude fish, mammal, cattle, goat, pig, sheep, rodent, hamster, mouse,rat, primate, and human; invertebrate animals include nematodes, otherworms, drosophila, and other insects.

The skilled artisan will appreciate that the enumerated organisms arealso useful for practicing other aspects of the invention, e.g., makingtransgenic organisms as described infra.

The cell having the target gene may be from the germ line or somatic,totipotent or pluripotent, dividing or non-dividing, parenchyma orepithelium, immortalized or transformed, or the like. The cell may be astem cell or a differentiated cell. Cell types that are differentiatedinclude adipocytes, fibroblasts, myocytes, cardiomyocytes, endothelium,neurons, glia, blood cells, megakaryocytes, lymphocytes, macrophages,neutrophils, eosinophils, basophils, mast cells, leukocytes,granulocytes, keratinocytes, chondrocytes, osteoblasts, osteoclasts,hepatocytes, and cells of the endocrine or exocrine glands.

Depending on the particular target gene and the dose of double strandedRNA material delivered, this process may provide partial or completeloss of function for the target gene. A reduction or loss of geneexpression in at least 50%, 60%, 70%, 80%, 90%, 95% or 99% or more oftargeted cells is exemplary. Inhibition of gene expression refers to theabsence (or observable decrease) in the level of protein and/or mRNAproduct from a target gene. Specificity refers to the ability to inhibitthe target gene without manifest effects on other genes of the cell. Theconsequences of inhibition can be confirmed by examination of theoutward properties of the cell or organism (as presented below in theexamples) or by biochemical techniques such as RNA solutionhybridization, nuclease protection, Northern hybridization, reversetranscription, gene expression monitoring with a microarray, antibodybinding, enzyme linked immunosorbent assay (ELISA), Western blotting,radioimmunoassay (RIA), other immunoassays, and fluorescence activatedcell analysis (FACS).

For RNA-mediated inhibition in a cell line or whole organism, geneexpression is conveniently assayed by use of a reporter or drugresistance gene whose protein product is easily assayed. Such reportergenes include acetohydroxyacid synthase (AHAS), alkaline phosphatase(AP), beta galactosidase (LacZ), beta glucoronidase (GUS),chloramphenicol acetyltransferase (CAT), green fluorescent protein(GFP), horseradish peroxidase (HRP), luciferase (Luc), nopaline synthase(NOS), octopine synthase (OCS), and derivatives thereof. Multipleselectable markers are available that confer resistance to ampicillin,bleomycin, chloramphenicol, gentarnycin, hygromycin, kanamycin,lincomycin, methotrexate, phosphinothricin, puromycin, and tetracyclin.Depending on the assay, quantitation of the amount of gene expressionallows one to determine a degree of inhibition which is greater than10%, 33%, 50%, 90%, 95% or 99% as compared to a cell not treatedaccording to the present invention. Lower doses of injected material andlonger times after administration of a RNA silencing agent (e.g., anRNAi agent) may result in inhibition in a smaller fraction of cells(e.g., at least 10%, 20%, 50%, 75%, 90%, or 95% of targeted cells).Quantitation of gene expression in a cell may show similar amounts ofinhibition at the level of accumulation of target mRNA or translation oftarget protein. As an example, the efficiency of inhibition may bedetermined by assessing the amount of gene product in the cell; mRNA maybe detected with a hybridization probe having a nucleotide sequenceoutside the region used for the inhibitory double-stranded RNA, ortranslated polypeptide may be detected with an antibody raised againstthe polypeptide sequence of that region.

The RNA may be introduced in an amount which allows delivery of at leastone copy per cell. Higher doses (e.g., at least 5, 10, 100, 500 or 1000copies per cell) of material may yield more effective inhibition; lowerdoses may also be useful for specific applications.

V. Methods of Treatment:

The present invention provides for both prophylactic and therapeuticmethods of treating a subject at risk of (or susceptible to) a diseaseor disorder or having a disease or disorder associated with aberrant orunwanted target gene expression or activity. The invention furtherprovides methods of treating cell or subject having (or susceptible to)an miRNA disorder.

“Treatment”, or “treating” as used herein, is defined as the applicationor administration of a therapeutic agent (e.g., a RNAi agent or agentmediating translational control, or vector or transgene encoding same)to a patient, or application or administration of a therapeutic agent toan isolated tissue or cell line from a patient, who has a disease ordisorder, a symptom of disease or disorder or a predisposition toward adisease or disorder, with the purpose to cure, heal, alleviate, relieve,alter, remedy, ameliorate, improve or affect the disease or disorder,the symptoms of the disease or disorder, or the predisposition towarddisease.

With regards to both prophylactic and therapeutic methods of treatment,such treatments may be specifically tailored or modified, based onknowledge obtained from the field of pharmacogenomics.“Pharmacogenomics”, as used herein, refers to the application ofgenomics technologies such as gene sequencing, statistical genetics, andgene expression analysis to drugs in clinical development and on themarket. More specifically, the term refers the study of how a patient'sgenes determine his or her response to a drug (e.g., a patient's “drugresponse phenotype”, or “drug response genotype”). Thus, another aspectof the invention provides methods for tailoring an individual'sprophylactic or therapeutic treatment with either the target genemolecules of the present invention or target gene modulators accordingto that individual's drug response genotype. Pharmacogenomics allows aclinician or physician to target prophylactic or therapeutic treatmentsto patients who will most benefit from the treatment and to avoidtreatment of patients who will experience toxic drug-related sideeffects.

1. Prophylactic Methods

In one aspect, the invention provides a method for preventing in asubject, a disease or condition associated with an aberrant or unwantedtarget gene expression or activity, by administering to the subject atherapeutic agent (e.g., a RNAi agent or agent mediating translationalcontrol, or vector or transgene encoding same). Subjects at risk for adisease which is caused or contributed to by aberrant or unwanted targetgene expression or activity can be identified by, for example, any or acombination of diagnostic or prognostic assays as described herein.Administration of a prophylactic agent can occur prior to themanifestation of symptoms characteristic of the target gene aberrancy,such that a disease or disorder is prevented or, alternatively, delayedin its progression. Depending on the type of target gene aberrancy, forexample, a target gene, target gene agonist or target gene antagonistagent can be used for treating the subject. The appropriate agent can bedetermined based on screening assays described herein.

2. Therapeutic Methods

Another aspect of the invention pertains to methods of modulating targetgene expression, protein expression or activity for therapeuticpurposes. Accordingly, in an exemplary embodiment, the modulatory methodof the invention involves contacting a cell capable of expressing targetgene with a therapeutic agent (e.g., a RNAi agent or agent mediatingtranslational control, or vector or transgene encoding same) that isspecific for the target gene or protein (e.g., is specific for the mRNAencoded by said gene or specifying the amino acid sequence of saidprotein) such that expression or one or more of the activities of targetprotein is modulated. These modulatory methods can be performed in vitro(e.g., by culturing the cell with the agent) or, alternatively, in vivo(e.g., by administering the agent to a subject). As such, the presentinvention provides methods of treating an individual afflicted with adisease or disorder characterized by aberrant or unwanted expression oractivity of a target gene polypeptide or nucleic acid molecule.Inhibition of target gene activity is desirable in situations in whichtarget gene is abnormally unregulated and/or in which decreased targetgene activity is likely to have a beneficial effect.

3. miRNA Therapy

Another aspect of the invention pertains to methods of treating a cellor subject having or susceptible to an miRNA disorder. mRNA disorderscan result, for example, when a naturally occurring miRNA is not presentin a cell or subject in an amount sufficient to regulate cellularprocesses controlled by the miRNA. In an exemplary embodiment, themethod of the invention involves administering to a subject having orsusceptible to an miRNA disorder an effective amount of a RNA silencingagent of the invention (e.g., a siRNA-like duplex) or vector ortransgene encoding same, the silencing agent delivering at least onestrand (e.g., a miRNA strand of the siRNA-like agent) to the subject(e.g., to the RISC complex of said subject). In another exemplaryembodiment, the method of the invention involves contacting a cell froma subject having or susceptible to an miRNA disorder with an effectiveamount of a RNA silencing agent of the invention (e.g., a siRNA-likeduplex) or vector or transgene encoding same, the silencing agentdelivering at least one strand (e.g., a miRNA strand of the siRNA-likeagent) to the cell (e.g., to the RISC complex of said cell). Aneffective amount of a siRNA-like agent is, for example, an amountsufficient to silence the natural target of the miRNA strand of thesiRNA-like duplex.

miRNAs have been shown to regulate a diverse set of biological processesin metazoans (e.g. invertebrates, vertebrates, and plants) includingcell proliferation and cell death (McManus M T et al., Semin. CancerBiol., 2003; Brenneke et al., Cell, 2003; Baehrecke E H. Curr. Biol.,2003), developmental timing (Pasquinelli A E and Ruvkun G, Annu Rev.Cell. Dev., 2002), embryonic and post-embryonic development (Lee R C andAmbros V, Science, 2001; Boutla A et al., Nuc. Acids. Res., 2003;Carrington J C and Ambros V, Science, 2003; Houbaviy H B et al., Dev.Cell, 2003; Aravin A A et al., Dev. Cell, 2003; Yekta et al., Science,2004; Vaucheret et al., Genes & Dev., 2004), neuronal development(Johnston R J and Hobert O, Nature, 2003; Kawasaki H and Taira K,Nature, 2003; Krichevsky A M et al., RNA, 2003; Kim J et al., PNAS,2004; Sempere L F et al., Genome Biology, 2004), fat metabolism (Xu etal., Curr. Biol., 2003), haematopoietic cell differentiation (Chen C Zet al, Science, 2004) and flower and leaf development (Aukerman M J andSakai H, Plant Cell, 2003; Bartel B and Bartel D P, Plant Physiol.,2003; Chen X, Science, 2003; Emery J F et al., Curr. Biol., 2003; Hakeet al., Curr. Biol., 2003; Hunter C and Poethig R, Curr. Opin. Genet.Dev., 2003; Kidner C A and Martienssen R A, Trends Genet., 2003;Palatnik J F, Nature, 2003; Achard P et al., Development, 2004; Zhong Ret al., Plant Cell Physiol., 2004; Kidner C A et al., Nature, 2004;Mallory A C et al., Curr. Biol., 2004). miRNA disorders can result insevere disruption of these processes. miRNA disorders have beenimplicated in a wide variety of plant and animal diseases includingcancer, neurological disorders (e.g. neurodegeneration), anddevelopmental defects. For example, miRNA disorders have been correlatedwith nonsmall cell lung cancer (Takamizawa et al., Cancer Research,2004), colorectal cancer (Michael M Z et al., Mol. Cancer. Res., 2003),chronic lymphocytic leukemia (Calin G A et al., PNAS, 2002; Calin G A etal., PNAS, 2004), Fragile X Syndrome (Caudy A et al, Genes & Dev.,2002), spinal muscular atrophy and Waisman Syndrome (Dostie et al., RNA,2003). Additional miRNA-silenced target gene linked with a particularmiRNA disorder can be identified in silico using computationalalgorithms described in the art (Rhoades et al. Cell, 2002; Enright etal. Genome Biol., 2003; Lewis et al., Cell, 2003; Stark et al., PLOSBiol., 2003).

The choice of which strand of the siRNA-like duplex is preferentiallyloaded in the RISC complex may depend on the nature of the miRNAdisorder. In one scenario, an miRNA disorders can result from adeficiency in the expression of a particular natural miRNA due to amutation or chromosomal lesion (e.g. translocation, inversion, ordeletion) in the miRNA gene or a regulatory sequence controlling itsexpression (e.g. an miRNA promoter). In another scenario, an miRNAdisorders can result from an acquisition of a mutation in a target mRNA.For example, a mutation in a miRNA-silenced target gene can render thetarget mRNA resistant to silencing, leading to overexpression of atarget mRNA that is normally silenced. Alternatively, a mutation in themiRNA-silenced target gene can render the target mRNA susceptible tomiRNA-guided cleavage, leading to underexpression of a target mRNA thatis normally constitutively expressed.

In another scenario, an miRNA disorder can result from the acquisitionof a mutation in a natural miRNA. A mutation in the 5′ end of an inindividual strand of an miRNA duplex can result in the loading the wrongstrand in RISC. For example, when the miRNA* strand is inappropriatelyloaded in RISC, the target mRNA may no longer be silenced, leading tooverexpression of a target mRNA that is normally silenced and/orunderexpression of a new, inappropriate target mRNA that is normally notsilenced (“off-target silencing”). In yet another scenario, an miRNAdisorder can result from a mutation in a gene encoding a component oftheRISC complex such that miRNA-mediated gene silencing is abrogated.

Use of the design rules described supra can abe applied to the RNAsilencing agents to correct for an miRNA disorder. In particular, miRNAduplex intermediates or siRNA-like duplexes that include a miRNA strandand/or a miRNA* strand can be altered with the design rules to promoteentry of the desired strand of the duplex (e.g., the miRNA strand) intoa RISC complex. In one aspect of the invention, entry of the miRNAstrand into the RISC complex is desired in order to treat the disorder.In another embodiment of the invention, entry of the miRNA* strand isdesired in order to treat the disorder. Entry of the miRNA strand may bepromoted by either lessening the base pair strength between the 5′ endof the miRNA strand and the 3′ end of the miRNA* strand (miRNA* 3′) ascompared to the base pair strength between the 3′ end of the miRNAstrand and the 5′ end of the miRNA* strand or enhancing the base pairstrength between the 3′ end of the miRNA strand and the 5′ end of themiRNA* strand as compared to the base pair strength between the 5′ endof miRNA strand and the 3′ end of the miRNA* strand. Alternatively,entry of the sense strand may be promoted by lessening the base pairstrength between the 5′ end of the miRNA* strand (miRNA* 5′) and the 3′end of the miRNA strand (miRNA 3′) as compared to the base pair strengthbetween the 3′ end of the miRNA* strand (miRNA* 3′) and the 5′ end ofthe miRNA strand (miRNA 5′) or enhancing the base pair strength betweenthe 3′ end of the miRNA* strand (miRNA 3′) and the 5′ end of the miRNAstrand (miRNA 5′) as compared to the base pair strength between the 5′end of miRNA* strand (miRNA* 5′) and the 3′ end of the miRNA strand(miRNA 3′).

4. Pharmacogenomics

The therapeutic agents (e.g., a RNAi agent or vector or transgeneencoding same) of the invention can be administered to individuals totreat (prophylactically or therapeutically) disorders associated withaberrant or unwanted target gene activity. In conjunction with suchtreatment, pharmacogenomics (i.e., the study of the relationship betweenan individual's genotype and that individual's response to a foreigncompound or drug) may be considered. Differences in metabolism oftherapeutics can lead to severe toxicity or therapeutic failure byaltering the relation between dose and blood concentration of thepharmacologically active drug. Thus, a physician or clinician mayconsider applying knowledge obtained in relevant pharmacogenomicsstudies in determining whether to administer a therapeutic agent as wellas tailoring the dosage and/or therapeutic regimen of treatment with atherapeutic agent.

Pharmacogenomics deals with clinically significant hereditary variationsin the response to drugs due to altered drug disposition and abnormalaction in affected persons. See, for example, Eichelbaum, M. et al.(1996) Clin. Exp. Pharmacol. Physiol. 23(10-11): 983-985 and Linder, M.W. et al. (1997) Clin. Chem. 43(2):254-266. In general, two types ofpharmacogenetic conditions can be differentiated. Genetic conditionstransmitted as a single factor altering the way drugs act on the body(altered drug action) or genetic conditions transmitted as singlefactors altering the way the body acts on drugs (altered drugmetabolism). These pharmacogenetic conditions can occur either as raregenetic defects or as naturally-occurring polymorphisms. For example,glucose-6-phosphate dehydrogenase deficiency (G6PD) is a commoninherited enzymopathy in which the main clinical complication ishaemolysis after ingestion of oxidant drugs (anti-malarials,sulfonamides, analgesics, nitrofurans) and consumption of fava beans.

One pharmacogenomics approach to identifying genes that predict drugresponse, known as “a genome-wide association”, relies primarily on ahigh-resolution map of the human genome consisting of already knowngene-related markers (e.g., a “bi-allelic” gene marker map whichconsists of 60,000-100,000 polymorphic or variable sites on the humangenome, each of which has two variants.) Such a high-resolution geneticmap can be compared to a map of the genome of each of a statisticallysignificant number of patients taking part in a Phase II/III drug trialto identify markers associated with a particular observed drug responseor side effect. Alternatively, such a high resolution map can begenerated from a combination of some ten-million known single nucleotidepolymorphisms (SNPs) in the human genome. As used herein, a “SNP” is acommon alteration that occurs in a single nucleotide base in a stretchof DNA. For example, a SNP may occur once per every 1000 bases of DNA. ASNP may be involved in a disease process, however, the vast majority maynot be disease-associated. Given a genetic map based on the occurrenceof such SNPs, individuals can be grouped into genetic categoriesdepending on a particular pattern of SNPs in their individual genome. Insuch a manner, treatment regimens can be tailored to groups ofgenetically similar individuals, taking into account traits that may becommon among such genetically similar individuals.

Alternatively, a method termed the “candidate gene approach”, can beutilized to identify genes that predict drug response. According to thismethod, if a gene that encodes a drugs target is known (e.g., a targetgene polypeptide of the present invention), all common variants of thatgene can be fairly easily identified in the population and it can bedetermined if having one version of the gene versus another isassociated with a particular drug response.

As an illustrative embodiment, the activity of drug metabolizing enzymesis a major determinant of both the intensity and duration of drugaction. The discovery of genetic polymorphisms of drug metabolizingenzymes (e.g., N-acetyltransferase 2 (NAT 2) and cytochrome P450 enzymesCYP2D6 and CYP2C19) has provided an explanation as to why some patientsdo not obtain the expected drug effects or show exaggerated drugresponse and serious toxicity after taking the standard and safe dose ofa drug. These polymorphisms are expressed in two phenotypes in thepopulation, the extensive metabolizer (EM) and poor metabolizer (PM).The prevalence of PM is different among different populations. Forexample, the gene coding for CYP2D6 is highly polymorphic and severalmutations have been identified in PM, which all lead to the absence offunctional CYP2D6. Poor metabolizers of CYP2D6 and CYP2C19 quitefrequently experience exaggerated drug response and side effects whenthey receive standard doses. If a metabolite is the active therapeuticmoiety, PM show no therapeutic response, as demonstrated for theanalgesic effect of codeine mediated by its CYP2D6-formed metabolitemorphine. The other extreme are the so called ultra-rapid metabolizerswho do not respond to standard doses. Recently, the molecular basis ofultra-rapid metabolism has been identified to be due to CYP2D6 geneamplification.

Alternatively, a method termed the “gene expression profiling”, can beutilized to identify genes that predict drug response. For example, thegene expression of an animal dosed with a therapeutic agent of thepresent invention can give an indication whether gene pathways relatedto toxicity have been turned on.

Information generated from more than one of the above pharmacogenomicsapproaches can be used to determine appropriate dosage and treatmentregimens for prophylactic or therapeutic treatment an individual. Thisknowledge, when applied to dosing or drug selection, can avoid adversereactions or therapeutic failure and thus enhance therapeutic orprophylactic efficiency when treating a subject with a therapeuticagent, as described herein.

Therapeutic agents can be tested in an appropriate animal model. Forexample, an RNAi agent (or expression vector or transgene encoding same)as described herein can be used in an animal model to determine theefficacy, toxicity, or side effects of treatment with said agent.Alternatively, a therapeutic agent can be used in an animal model todetermine the mechanism of action of such an agent. For example, anagent can be used in an animal model to determine the efficacy,toxicity, or side effects of treatment with such an agent.Alternatively, an agent can be used in an animal model to determine themechanism of action of such an agent.

VI. Pharmaceutical Compositions

The invention pertains to uses of the above-described agents fortherapeutic treatments as described infra. Accordingly, the modulatorsof the present invention can be incorporated into pharmaceuticalcompositions suitable for administration. Such compositions typicallycomprise the nucleic acid molecule, protein, antibody, or modulatorycompound and a pharmaceutically acceptable carrier. As used herein thelanguage “pharmaceutically acceptable carrier” is intended to includeany and all solvents, dispersion media, coatings, antibacterial andantifungal agents, isotonic and absorption delaying agents, and thelike, compatible with pharmaceutical administration. The use of suchmedia and agents for pharmaceutically active substances is well known inthe art. Except insofar as any conventional media or agent isincompatible with the active compound, use thereof in the compositionsis contemplated. Supplementary active compounds can also be incorporatedinto the compositions.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, intraperitoneal, intramuscular, oral (e.g., inhalation),transdermal (topical), and transmucosal administration. Solutions orsuspensions used for parenteral, intradermal, or subcutaneousapplication can include the following components: a sterile diluent suchas water for injection, saline solution, fixed oils, polyethyleneglycols, glycerine, propylene glycol or other synthetic solvents;antibacterial agents such as benzyl alcohol or methyl parabens;antioxidants such as ascorbic acid or sodium bisulfite; chelating agentssuch as ethylenediaminetetraacetic acid; buffers such as acetates,citrates or phosphates and agents for the adjustment of tonicity such assodium chloride or dextrose. pH can be adjusted with acids or bases,such as hydrochloric acid or sodium hydroxide. The parenteralpreparation can be enclosed in ampoules, disposable syringes or multipledose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyetheylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle which containsa basic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying which yields a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g.,with conventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD50/ED50.Compounds that exhibit large therapeutic indices are preferred. Althoughcompounds that exhibit toxic side effects may be used, care should betaken to design a delivery system that targets such compounds to thesite of affected tissue in order to minimize potential damage touninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED50 with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose may beformulated in animal models to achieve a circulating plasmaconcentration range that includes the EC50 (i.e., the concentration ofthe test compound which achieves a half-maximal response) as determinedin cell culture. Such information can be used to more accuratelydetermine useful doses in humans. Levels in plasma may be measured, forexample, by high performance liquid chromatography.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

VI. Knockout and/or Knockdown Cells or Organisms

A further preferred use for the RNAi agents of the present invention (orvectors or transgenes encoding same) is a functional analysis to becarried out in eukaryotic cells, or eukaryotic non-human organisms,preferably mammalian cells or organisms and most preferably human cells,e.g. cell lines such as HeLa or 293 or rodents, e.g. rats and mice. Byadministering a suitable RNAi agent which is sufficiently complementaryto a target mRNA sequence to direct target-specific RNA interference, aspecific knockout or knockdown phenotype can be obtained in a targetcell, e.g. in cell culture or in a target organism.

Thus, a further subject matter of the invention is a eukaryotic cell ora eukaryotic non-human organism exhibiting a target gene-specificknockout or knockdown phenotype comprising a fully or at least partiallydeficient expression of at least one endogeneous target gene whereinsaid cell or organism is transfected with at least one vector comprisingDNA encoding an RNAi agent capable of inhibiting the expression of thetarget gene. It should be noted that the present invention allows atarget-specific knockout or knockdown of several different endogeneousgenes due to the specificity of the RNAi agent.

Gene-specific knockout or knockdown phenotypes of cells or non-humanorganisms, particularly of human cells or non-human mammals may be usedin analytic to procedures, e.g. in the functional and/or phenotypicalanalysis of complex physiological processes such as analysis of geneexpression profiles and/or proteomes. Preferably the analysis is carriedout by high throughput methods using oligonucleotide based chips.

Using RNAi based knockout or knockdown technologies, the expression ofan endogeneous target gene may be inhibited in a target cell or a targetorganism. The endogeneous gene may be complemented by an exogenoustarget nucleic acid coding for the target protein or a variant ormutated form of the target protein, e.g. a gene or a DNA, which mayoptionally be fused to a further nucleic acid sequence encoding adetectable peptide or polypeptide, e.g. an affinity tag, particularly amultiple affinity tag.

Variants or mutated forms of the target gene differ from the endogeneoustarget gene in that they encode a gene product which differs from theendogeneous gene product on the amino acid level by substitutions,insertions and/or deletions of single or multiple amino acids. Thevariants or mutated forms may have the same biological activity as theendogeneous target gene. On the other hand, the variant or mutatedtarget gene may also have a biological activity, which differs from thebiological activity of the endogeneous target gene, e.g. a partiallydeleted activity, a completely deleted activity, an enhanced activityetc. The complementation may be accomplished by compressing thepolypeptide encoded by the endogeneous nucleic acid, e.g. a fusionprotein comprising the target protein and the affinity tag and thedouble stranded RNA molecule for knocking out the endogeneous gene inthe target cell. This compression may be accomplished by using asuitable expression vector expressing both the polypeptide encoded bythe endogenous nucleic acid, e.g. the tag-modified target protein andthe double stranded RNA molecule or alternatively by using a combinationof expression vectors. Proteins and protein complexes which aresynthesized de novo in the target cell will contain the exogenous geneproduct, e.g., the modified fusion protein. In order to avoidsuppression of the exogenous gene product by the RNAi agent, thenucleotide sequence encoding the exogenous nucleic acid may be alteredat the DNA level (with or without causing mutations on the amino acidlevel) in the part of the sequence which is homologous to the RNAiagent. Alternatively, the endogeneous target gene may be complemented bycorresponding nucleotide sequences from other species, e.g. from mouse.

VII. Transgenic Organisms

Engineered RNA precursors of the invention can be expressed intransgenic animals. These animals represent a model system for the studyof disorders that are caused by, or exacerbated by, overexpression orunderexpression (as compared to wildtype or normal) of nucleic acids(and their encoded polypeptides) targeted for destruction by the RNAiagents, e.g., siRNAs and shRNAs, and for the development of therapeuticagents that modulate the expression or activity of nucleic acids orpolypeptides targeted for destruction.

Transgenic animals can be farm animals (pigs, goats, sheep, cows,horses, rabbits, and the like), rodents (such as rats, guinea pigs, andmice), non-human primates (for example, baboons, monkeys, andchimpanzees), and domestic animals (for example, dogs and cats).Invertebrates such as Caenorhabditis elegans or Drosophila can be usedas well as non-mammalian vertebrates such as fish (e.g., zebrafish) orbirds (e.g., chickens).

Engineered RNA precursors with stems of 18 to 30 nucleotides in lengthare preferred for use in mammals, such as mice. A transgenic founderanimal can be identified based upon the presence of a transgene thatencodes the new RNA precursors in its genome, and/or expression of thetransgene in tissues or cells of the animals, for example, using PCR orNorthern analysis. Expression is confirmed by a decrease in theexpression (RNA or protein) of the target sequence.

A transgenic founder animal can be used to breed additional animalscarrying the transgene. Moreover, transgenic animals carrying atransgene encoding the RNA precursors can further be bred to othertransgenic animals carrying other transgenes. In addition, cellsobtained from the transgenic founder animal or its offspring can becultured to establish primary, secondary, or immortal cell linescontaining the transgene.

1. Procedures for Making Transgenic, Non-Human Animals

A number of methods have been used to obtain transgenic, non-humananimals, which are animals that have gained an additional gene by theintroduction of a transgene into their cells (e.g., both the somatic andgenn cells), or into an ancestor's genn line. In some cases, transgenicanimals can be generated by commercial facilities (e.g., The TransgenicDrosophila Facility at Michigan State University, The TransgenicZebrafish Core Facility at the Medical College of Georgia (Augusta,Ga.), and Xenogen Biosciences (St. Louis, Mo.). In general, theconstruct containing the transgene is supplied to the facility forgenerating a transgenic animal.

Methods for generating transgenic animals include introducing thetransgene into the germ line of the animal. One method is bymicroinjection of a gene construct into the pronucleus of an early stageembryo (e.g., before the four-cell stage; Wagner et al., 1981, Proc.Natl. Acad. Sci. USA 78:5016; Brinster et al., 1985, Proc. Natl. Acad.Sci. USA 82:4438). Alternatively, the transgene can be introduced intothe pronucleus by retroviral infection. A detailed procedure forproducing such transgenic mice has been described (see e.g., Hogan etal., MP1 ulating the Mouse ErnbnLo. Cold Spring Harbour Laboratory, ColdSpring Harbour, N.Y. (1986); U.S. Pat. No. 5,175,383 (1992)). Thisprocedure has also been adapted for other animal species (e.g., Hammeret al., 1985, Nature 315:680; Murray et al., 1989, Reprod. Fert. Devl.1:147; Pursel et al., 1987, Vet. Immunol. Histopath. 17:303; Rexroad etal., 1990, J. Reprod. Fert. 41 (suppl): 1 19; Rexroad et al., 1989,Molec. Reprod. Devl. 1:164; Simons et al., 1988, BioTechnology 6:179;Vize et al., 1988, J. Cell. Sci. 90:295; and Wagner, 1989, J. Cell.Biochem. 13B (suppl): 164).

In brief, the procedure involves introducing the transgene into ananimal by microinjecting the construct into the pronuclei of thefertilized mammalian egg(s) to cause one or more copies of the transgeneto be retained in the cells of the developing mammal(s). Followingintroduction of the transgene construct into the fertilized egg, the eggmay be incubated in vitro for varying amounts of time, or reimplanted ain surrogate host, or both. One common method is to incubate the embryosin vitro for about 1-7 days, depending on the species, and thenreimplant them into the surrogate host. The presence of the transgene inthe progeny of the transgenically manipulated embryos can be tested bySouthern blot analysis of a segment of tissue.

Another method for producing germ-line transgenic animals is through theuse of embryonic stem (ES) cells. The gene construct can be introducedinto embryonic stem cells by homologous recombination (Thomas et al.,1987, Cell 51:503; Capecchi, Science 1989, 244:1288; Joyner et al.,1989, Nature 338:153) in a transcriptionally active region of thegenome. A suitable construct can also be introduced into embryonic stemcells by DNA-mediated transfection, such as by 17 electroporation(Ausubel et al., Current Protocols in Molecular Biology, John Wiley &Sons, 1987). Detailed procedures for culturing embryonic stem cells(e.g., ES-D3@ ATCC# CCL-1934, ES-E14TG2a, ATCC# CCL-1821, American TypeCulture Collection, Rockville, AM) and methods of making transgenicanimals from embryonic stem cells can be found in Teratocarcinomas andEmbryonic Stem Cells, A Practical Approach, ed. E. J. Robertson (IRLPress, 1987). In brief, the ES cells are obtained from pre-implantationembryos cultured in vitro (Evans et al., 1981, Nature 292:154-156).Transgenes can be efficiently introduced into ES cells by DNAtransfection or by retrovirus-mediated transduction. The resultingtransformed ES cells can thereafter be combined with blastocysts from anon-human animal. The ES cells colonize the embryo and contribute to thegenn line of the resulting chimeric animal.

In the above methods, the transgene can be introduced as a linearconstruct, a circular plasmid, or a viral vector, which can beincorporated and inherited as a transgene integrated into the hostgenome. The transgene can also be constructed to permit it to beinherited as an extrachromosomal plasmid (Gassmann et al., 1995, Proc.Natl. Acad. Sci. USA 92:1292). A plasmid is a DNA molecule that canreplicate autonomously in a host.

The transgenic, non-human animals can also be obtained by infecting ortransfecting cells either in vivo (e.g., direct injection), ex vivo(e.g., infecting the cells outside the host and later reimplanting), orin vitro (e.g., infecting the cells outside host), for example, with arecombinant viral vector carrying a gene encoding the engineered RNAprecursors. Examples of suitable viral vectors include recombinantretroviral vectors (Valerio et al., 1989, Gene 84:419; Scharfinan etal., 1991, Proc. Natl. Acad. Sci. USA 88:462; Miller and Buttimore,1986, Mol. Cell. Biol. 6:2895), recombinant adenoviral vectors (Freidmanet al., 1986, Mol. Cell. Biol. 6:3791; Levrero et al., 1991, Gene 101:195), and recombinant Herpes simplex viral vectors (Fink et al., 1992,Human Gene Therapy 3:11). Such methods are also useful for introducingconstructs into cells for uses other than generation of transgenicanimals.

Other approaches include insertion of transgenes encoding the newengineered RNA precursors into viral vectors including recombinantadenovirus, adenoassociated virus, and herpes simplex virus-1, orrecombinant bacterial or eukaryotic plasmids. Viral vectors transfectcells directly. Other approaches include delivering the transgenes, inthe form of plasmid DNA, with the help of, for example, cationicliposomes (lipofectin) or derivatized (e.g. antibody conjugated)polylysine conjugates, gramacidin S, artificial viral envelopes, orother such intracellular carriers, as well as direct injection of thetransgene construct or CaPO₄ precipitation carried out in vivo. Suchmethods can also be used in vitro to introduce constructs into cells foruses other than generation of transgenic animals.

Retrovirus vectors and adeno-associated virus vectors can be used as arecombinant gone delivery system for the transfer of exogenous genes invivo or in vitro. These vectors provide efficient delivery of genes intocells, and the transferred nucleic acids are stably integrated into thechromosomal DNA of the host. The development of specialized cell lines(termed “packaging cells”) which produce only replication-defectiveretroviruses has increased the utility of retroviruses for gene therapy,and defective retroviruses are characterized for use in gene transferfor gene therapy purposes (for a review see Miller, 1990, Blood 76:271).A replication defective retrovirus can be packaged into virions whichcan be used to infect a target cell through the use of a helper virus bystandard techniques. Protocols for producing recombinant retrovirusesand for infecting cells in vitro or in vivo with such viruses can befound in Current Protocols in Molecular Biology, Ausubel, F. M. et al.,(eds.) Greene Publishing Associates, (1989), Sections 9 9.14 and otherstandard laboratory manuals.

Examples of suitable retroviruses include pLJ, pZIP, pWE and pEM whichare known to those skilled in the art. Examples of suitable packagingvirus lines for preparing both ecotropic and amphotropic retroviralsystems include Psi-Crip, PsiCre, Psi-2 and Psi-Am. Retroviruses havebeen used to introduce a variety of genes into many different celltypes, including epithelial cells, in vitro and/or in vivo (see forexample Eglitis, et al., 1985, Science 230:1395-1398; Danos andMulligan, 1988, Proc. Natl. Acad. Sci. USA 85:6460-6464; Wilson et al.,1988, Proc. Natl. Acad. Sci. USA 85:3014-3018; Armentano et al., 1990,Proc. Natl. Acad. Sci. USA 87:6141-6145; Huber et al., 1991, Proc. Natl.Acad. Sci. USA 88:8039-8043; Ferry et al., 1991, Proc. Natl. Acad. Sci.USA 88:8377-8381; Chowdhury et al., 1991, Science 254:1802-1805; vanBeusechem. et al., 1992, Proc. Nad. Acad. Sci. USA 89:7640-19; Kay etal., 1992, Human Gene Therapy 3:641-647; Dai et al., 1992, Proc. Natl.Acad. Sci. USA 89:10892-10895; Hwu et al., 1993, J. Immunol.150:4104-4115; U.S. Pat. No. 4,868,116; U.S. Pat. No. 4,980,286; PCTApplication WO 89/07136; PCT Application WO 89/02468; PCT Application WO89/05345; and PCT Application WO 92/07573).

In another example, recombinant retroviral vectors capable oftransducing and expressing genes inserted into the genome of a cell canbe produced by transfecting the recombinant retroviral genome intosuitable packaging cell lines such as PA317 and Psi-CRIP (Comette etal., 1991, Human Gene Therapy 2:5-10; Cone et al., 1984, Proc. Natl.Acad. Sci. USA 81:6349). Recombinant adenoviral vectors can be used toinfect a wide variety of cells and tissues in susceptible hosts (e.g.,rat, hamster, dog, and chimpanzee) (Hsu et al., 1992, J. InfectiousDisease, 166:769), and also have the advantage of not requiringmitotically active cells for infection. Another viral gene deliverysystem useful in the present invention also utilizes adenovirus-derivedvectors. The genome of an adenovirus can be manipulated such that itencodes and expresses a gene product of interest but is inactivated interms of its ability to replicate in a normal lytic viral life cycle.See, for example, Berkner et al. (1988, BioTechniques 6:616), Rosenfeldet al. (1991, Science 252:431-434), and Rosenfeld et al. (1992, Cell68:143-155). Suitable adenoviral vectors derived from the adenovirusstrain Ad type 5 d1324 or other strains of adenovirus (e.g., Ad2, AO,Ad7 etc.) are known to those skilled in the art.

Recombinant adenoviruses can be advantageous in certain circumstances inthat they are not capable of infecting nondividing cells and can be usedto infect a wide variety of cell types, including epithelial cells(Rosenfeld et al., 1992, cited supra). Furthernore, the virus particleis relatively stable and amenable to purification and concentration, andas above, can be modified to affect the spectrum of infectivity.Additionally, introduced adenoviral DNA (and foreign DNA containedtherein) is not integrated into the genome of a host cell but remainsepisomal, thereby avoiding potential problems that can occur as a resultof insertional mutagenesis hz situ where introduced DNA becomesintegrated into the host genome (e.g., retroviral DNA). Moreover, thecarrying capacity of the adenoviral genome for foreign DNA is large (upto 8 kilobases) relative to other gene delivery vectors (Berkner et al.cited supra; Haj-Ahmand and Graham, 1986, J. Virol. 57:267).

Yet another viral vector system useful for delivery of the subjecttransgenes is the adeno-associated virus (AAV). Adeno-associated virusis a naturally occurring defective virus that requires another virus,such as an adenovirus or a herpes virus, as a helper virus for efficientreplication and a productive life cycle. For a review, see Muzyczka etal. (1992, Curr. Topics in Micro. and Immunol. 158:97-129). It is alsoone of the few viruses that may integrate its DNA into non-dividingcells, and exhibits a high frequency of stable integration (see forexample Flotte et al. (1992, Am. J. Respir. Cell. Mol. Biol. 7:349-356;Samulski et al., 1989, J. Virol. 63:3822-3828; and McLaughlin et al.(1989, J. Virol. 62:1963-1973). Vectors containing as little as 300 basepairs of AAV can be packaged and can integrate. Space for exogenous DNAis limited to about 4.5 kb. An AAV vector such as that described inTratschin et al. (1985) Mol. Cell. Biol. 5:3251-3260 can be used tointroduce DNA into cells. A variety of nucleic acids have beenintroduced into different cell types using AAV vectors (see for exampleHennonat et al. (1984) Proc. Nad. Acad. Sci. USA 8 1:6466-6470;Tratschin et al. (1985) Mol. Cell. Biol. 4:2072-2081; Wondisford et al.(1988) Mol. Endocrinol. 2:32-39; Tratschin et al. (1984) J. Virol.51:611-619; and Flotte et al. (1993) J. Biol. Chem. 268:3781-3790).

In addition to viral transfer methods, such as those illustrated above,non-viral methods can also be employed to cause expression of an shRNAor engineered RNA precursor of the invention in the tissue of an animal.Most non-viral methods of gene transfer rely on normal mechanisms usedby mammalian cells for the uptake and intracellular transport ofmacromolecules. In preferred embodiments, non-viral gene deliverysystems of the present invention rely on endocytic pathways for theuptake of the subject gene of the invention by the targeted cell.Exemplary gene delivery systems of this type include liposomal derivedsystems, poly-lysine conjugates, and artificial viral envelopes. Otherembodiments include plasmid injection systems such as are described inMeuli et al., (2001) J. Invest. DerinatoL, 116(1):131-135; Cohen et al.,(2000) Gene Ther., 7(22):1896-905; and Tam et al., (2000) Gene Ther.,7(21):186774.

In a representative embodiment, a gene encoding an shRNA or engineeredRNA precursor of the invention can be entrapped in liposomes bearingpositive charges on their surface (e.g., lipofectins) and (optionally)which are tagged with antibodies against cell surface antigens of thetarget tissue (Mizuno et al., (1992) No Shinkei Geka, 20:547-55 1; PCTpublication WO91/06309; Japanese patent application 10473 8 1; andEuropean patent publication EP-A-43 075).

Animals harboring the transgene can be identified by detecting thepresence of the transgene in genomic DNA (e.g., using Southernanalysis). In addition, expression of the shRNA or engineered RNAprecursor can be detected directly (e.g., by Northern analysis).Expression of the transgene can also be confirmed by detecting adecrease in the amount of protein corresponding to the targetedsequence. When the transgene is under the control of an inducible ordevelopmentally regulated promoter, expression of the target protein isdecreased when the transgene is induced or at the developmental stagewhen the transgene is expressed, respectively.

2. Clones of Transgenic Animals

Clones of the non-human transgenic animals described herein can beproduced according to the methods described in Wilmut et al. ((1997)Nature, 385:810-813) and PCT publication Nos. WO 97/07668 and WO97/07669. In brief, a cell, e.g., a somatic cell from the transgenicanimal, can be isolated and induced to exit the growth cycle and enterthe G0 phase to become quiescent. The quiescent cell can then be fused,e.g., through the use of electrical pulses, to an enucleated oocyte froman animal of the same species from which the quiescent cell is isolated.The reconstructed oocyte is then cultured such that it develops into amorula or blastocyte and is then transferred to a pseudopregnant femalefoster animal. Offspring borne of this female foster animal will beclones of the animal from which the cell, e.g., the somatic cell, wasisolated.

Once the transgenic animal is produced, cells of the transgenic animaland cells from a control animal are screened to determine the presenceof an RNA precursor nucleic acid sequence, e.g., using polymerase chainreaction (PCR). Alternatively, the cells can be screened to determine ifthe RNA precursor is expressed (e.g., by standard procedures such asNorthern blot analysis or reverse transcriptase-polymerase chainreaction (RT-PCR); Sambrook et al., Molecular Cloning—A LaboratoryManual, (Cold Spring Harbor Laboratory, 1989)).

The transgenic animals of the present invention can be homozygous orheterozygous, and one of the benefits of the invention is that thetarget mRNA is effectively degraded even in heterozygotes. The presentinvention provides for transgenic animals that carry a transgene of theinvention in all their cells, as well as animals that carry a transgenein some, but not all of their cells. That is, the invention provides formosaic animals. The transgene can be integrated as a single transgene orin concatatners, e.g., head-to-head tandems or head-to-tail tandems.

For a review of techniques that can be used to generate and assesstransgenic animals, skilled artisans can consult Gordon (IwL Rev. CytoL1 1 5:171-229, 1989), and may obtain additional guidance from, forexample: Hogan et al. “Manipulating the Mouse Embryo” (Cold SpringHarbor Press, Cold Spring Harbor, N.Y., 1986; Krimpenfort et al.,Bio/Technology 9:86, 1991; Palmiter et al., Cell 41:343, 1985; Kraemeret al., “Genetic Manipulation of the Early Mammalian Embryo,” ColdSpring Harbor Press, Cold Spring Harbor, N.Y., 1985; Hammer et al.,Nature 315:680, 1985; Purcel et al., Scieizce, 244:1281, 1986; Wagner etal., U.S. Pat. No. 5,175,385; and Krimpenfort et al., U.S. Pat. No.5,175,384.

3. Transgenic Plants

Among the eukaryotic organisms featured in the invention are plantscontaining an exogenous nucleic acid that encodes an engineered RNAprecursor of the invention.

Accordingly, a method according to the invention comprises making aplant having a nucleic acid molecule or construct, e.g., a transgene,described herein. Techniques for introducing exogenous micleic acidsinto monocotyledonous and dicotyledonous plants are known in the art,and include, without limitation, Agrobacterium-mediated transformation,viral vector-mediated transformation, electroporation and particle guntransformation, see, e.g., U.S. Pat. Nos. 5,204,253 and 6,013,863. If acell or tissue culture is used as the recipient tissue fortransformation, plants can be regenerated from transformed cultures bytechniques known to those skilled in the art. Transgenic plants can beentered into a breeding program, e.g., to introduce a nucleic acidencoding a polypeptide into other lines, to transfer the nucleic acid toother species or for further selection of other desirable traits.Alternatively, transgenic plants can be propagated vegetatively forthose species amenable to such techniques. Progeny includes descendantsof a particular plant or plant line. Progeny of a plant include seedsformed on F1, F2, F3, and subsequent generation plants, or seeds formedon BQ, BC2, BC3, and subsequent generation plants. Seeds produced by atransgenic plant can be grown and then selfed (or outcrossed and selfed)to obtain seeds homozygous for the nucleic acid encoding a novelpolypeptide.

A suitable group of plants with which to practice the invention includedicots, such as safflower, alfalfa, soybean, rapeseed (high erucic acidand canola), or sunflower. Also suitable are monocots such as corn,wheat, rye, barley, oat, rice, millet, amaranth or sorghum. Alsosuitable are vegetable crops or root crops such as potato, broccoli,peas, sweet corn, popcorn, tomato, beans (including kidney beans, limabeans, dry beans, green beans) and the like. Also suitable are fruitcrops such as peach, pear, apple, cherry, orange, lemon, grapefruit,plum, mango and palm. Thus, the invention has use over a broad range ofplants, including species from the genera Anacardium, Arachis,Asparagus, Atropa, Avena, Brassica, Citrus, Citrullus, Capsicum,Carthamus, Cocos, Coffea, Cucumis, Cucurbita, Daucus, Elaeis, Fragaria,Glycine, Gossypium, Helianthus, Heterocallis, Hordeum, Hyoscyalnus,Lactuca, Linum, Lolium, Lupinus, Lycopersicon, Malus, Manihot, Majorana,Medicago, Nicotiana, Olea, Oryza, Panicum, Pannesetum, Persea,Phaseolus, Pistachia, Pisum, Pyrus, Prunus, Raphanus, Ricinus, Secale,Senecio, Sinapis, Solanum, Sorghum, Theobromus, Trigonella, Triticum,Vicia, Vitis, Vigna and Zea.

The skilled artisan will appreciate that the enumerated organisms arealso useful for practicing other aspects of the invention, e.g., as hostcells, as described supra.

The nucleic acid molecules of the invention can be expressed in plantsin a cell- or tissue-specific manner according to the regulatoryelements chosen to include in a particular nucleic acid constructpresent in the plant. Suitable cells, tissues, and organs in which toexpress a chimeric polypeptide of the invention include, withoutlimitation, egg cell, central cell, synergid cell, zygote, ovuleprimordia, nucellus, integuments, endothelium, female garnetophytecells, embryo, axis, cotyledons, suspensor, endosperm, seed coat, groundmeristem, vascular bundle, cambium, phloem, cortex, shoot or root apicalmeristems, lateral shoot or root meristems, floral meristem, leafprimordia, leaf mesophyll cells, and leaf epidermal cells, e.g.,epidermal cells involved in fortning the cuticular layer. Also suitableare cells and tissues grown in liquid media or on semi-solid media.

4. Transgenic Fungi

Other eukaryotic organisms featured in the invention are fungicontaining an exogenous nucleic acid molecule that encodes an engineeredRNA precursor of the invention. Accordingly, a method according to theinvention comprises introducing a nucleic acid molecule or construct asdescribed herein into a fungus. Techniques for introducing exogenousnucleic acids into many fungi are known in the art, see, e.g., U.S. Pat.Nos. 5,252,726 and 5,070,020. Transformed fungi can be cultured bytechniques known to those skilled in the art. Such fungi can be used tointroduce a nucleic acid encoding a polypeptide into other fungalstrains, to transfer the nucleic acid to other species or for furtherselection of other desirable traits.

A suitable group of fungi with which to practice the invention includefission yeast and budding yeast, such as Saccharoinyces cereviseae, S.pombe, S. carlsbergeris and Candida albicans. Filamentous fungi such asAspergillus spp. and Penicillium spp. are also useful.

VIII. Functional Genomics and/or Proteomics

Preferred applications for the cell or organism of the invention is theanalysis of gene expression profiles and/or proteomes. In an especiallypreferred embodiment an analysis of a variant or mutant form of one orseveral target proteins is carried out, wherein said variant or mutantforms are reintroduced into the cell or organism by an exogenous targetnucleic acid as described above. The combination of knockout of anendogeneous gene by application of a RNA silencing agent of theinvention and rescue of the knockout by using mutated, e.g. partiallydeleted exogenous target has advantages compared to the use of aknockout cell. Further, this method is particularly suitable foridentifying functional domains of the targeted protein. In a furtherpreferred embodiment a comparison, e.g. of gene expression profilesand/or proteomes and/or phenotypic characteristics of at least two cellsor organisms is carried out. These organisms are selected from: (i) acontrol cell or control organism without target gene inhibition, (ii) acell or organism with target gene inhibition and (iii) a cell ororganism with target gene inhibition plus target gene complementation byan exogenous target nucleic acid.

Furthermore, the RNA knockout complementation method may be used for ispreparative purposes, e.g. for the affinity purification of proteins orprotein complexes from eukaryotic cells, particularly mammalian cellsand more particularly human cells. In this embodiment of the invention,the exogenous target nucleic acid preferably codes for a target proteinwhich is fused to art affinity tag. This method is suitable forfunctional proteome analysis in mammalian cells, particularly humancells.

Another utility of the present invention could be a method ofidentifying gene function in a cell or organism comprising the use of anRNA silencing agent of the invention (e.g. RNAi agent or agent causingtranslational repression) to inhibit the activity of a target gene ofpreviously unknown function. Instead of the time consuming and laboriousisolation of mutants by traditional genetic screening, functionalgenomics would envision determining the function of uncharacterizedgenes by employing the RNA silencing agents of the invention to reducethe amount and/or alter the timing of target gene activity.

The invention could be used in determining potential pharmaceutics,potential targets for pharmaceutics, understanding normal andpathological events associated with development, determining signalingpathways responsible for postnatal development/aging, and the like. Theincreasing speed of acquiring nucleotide sequence information fromgenomic and expressed gene sources, including total genome sequences formodel organisms (e.g. rat, mouse, zebrafish, yeast, Arabidopsisthalania, D. melanogaster, and C. elegans), can be coupled with theinvention to determine gene function in that organisms, a relatedorganism (e.g., a parasitic fly or nematode), or an organism containinga homolog of the gene of interest (e.g. human). The preference ofdifferent organisms to use particular codons, searching sequencedatabases for related gene products, correlating the linkage map ofgenetic traits with the physical map from which the nucleotide sequencesare derived, and artificial intelligence methods may be used to defineputative open reading frames from the nucleotide sequences acquired insuch sequencing projects. A simple assay would be to inhibit geneexpression according to the partial sequence available from an expressedsequence tag (EST). Functional alterations in growth, development,metabolism, disease resistance, or other biological processes would beindicative of the normal role of the EST's gene product.

The ease with which RNA can be introduced into an intact cell or modelorganism containing the target gene allows the RNA silencing agents ofthe present invention to be used in high throughput screening (HTS).Solutions containing RNA silencing agents (e.g. RNAi agents ortranslational repression agents) that are capable of inhibiting thedifferent expressed genes can be placed into individual wells positionedon a microtiter plate as an ordered array, and intact cells organisms ineach well can be assayed for any changes or modifications in behavior ordevelopment due to inhibition of target gene activity. The amplified RNAcan be fed directly to, injected into, the cell/organism containing thetarget gene. Alternatively, the RNA silencing agent (e.g. RNAi agent ortranslational repression agent) can be produced from a vector, asdescribed herein. Vectors can be injected into the cell/organismcontaining the target gene. The function of the target gene can beassayed from the effects it has on the cell/organism when gene activityis inhibited. This screening could be amenable to small subjects thatcan be processed in large number, for example: arabidopsis, bacteria,drosophila, fungi, nematodes, viruses, zebrafish, and tissue culturecells derived from mammals. A nematode or other organism that produces acolorimetric, fluorogenic, or luminescent signal in response to aregulated promoter (e.g., transfected with a reporter gene construct)can be assayed in an HTS format.

The present invention may be useful in allowing the inhibition ofessential genes. Such genes may be required for cell or organismviability at only particular stages of development or cellularcompartments. The functional equivalent of conditional mutations may beproduced by inhibiting activity of the target gene when or where it isnot required for viability. The invention allows addition of the RNAsilencing agent (e.g. RNAi or translational repression agent) atspecific times of development and locations in the organism withoutintroducing permanent mutations into the target genome.

siRNA-like silencing agents of the present invention can be used inmethod for identifying the natural target (e.g., the natural mRNAtarget) of a miRNA in a cell or organism. An exemplary method comprisescontacting a plurality or panel of candidate target genes (e.g., targetmRNA sequences), for example, several hundred natural mRNAs representedon a microwell plate). mRNAs can be separated into individual wellspositioned on a microtiter plate as an ordered array. The miRNA can thenbe assessed for its ability to bind to, direct cleavage of, recruit RISCcomponents, or any other indicator of RNA silencing (e.g., by RNAi ortranslational repression) using methods described herein or routine inthe art. In other embodiments, siRNA-like duplexes or miRNAs can bearrayed on plates for testing with a specific target gene or mRNA. Inpreferred embodiments, the miRNAs are used at low concentrations (e.g.picomolar concentration) to prevent non-specific silencing of thetarget.

When used to analyse gene expression profiles and/or proteomes, toidentify or inhibit gene function, or to validate pharmaceutic targets,the RNA silencing agents of the invention offer improvements overexisting RNA silencing agents. The RNA silencing agents of the inventionhave several-fold improvement in potency (e.g., can be used at picomolarconcentrations vs. the nanomolar or micromolar concentrations requiredwhen using un-modified, e.g., symmetrical silencing agents) so thatlower and more economical amounts of these reagents can be used for themethods described supra. Alternatively, the use of the RNA silencingagents of the invention may obviate the need for costly chemicalmodifications.

IX. Screening Assays

The methods of the invention are also suitable for use in methods toidentify and/or characterize potential pharmacological agents, e.g.identifying new pharmacological agents from a collection of testsubstances and/or characterizing mechanisms of action and/or sideeffects of known pharmacological agents. In one exemplary embodiment, alibrary of pharmaceutical agents can be screened to identify apharmaceutical agent which suppresses or “rescues” the gene silencingphenotype in a cell or organism caused by previous administration of theRNA silencing agent.

Thus, the present invention also relates to a system for identifyingand/or characterizing pharmacological agents acting on at least onetarget protein comprising: (a) a eukaryotic cell or a eukaryoticnon-human organism capable of expressing at least one endogeneous targetgene coding for said so target protein, (b) at least one silencing agent(e.g. RNAi agent) molecule capable of inhibiting the expression of saidat least one endogeneous target gene, and (c) a test substance or acollection of test substances wherein pharmacological properties of saidtest substance or said collection are to be identified and/orcharacterized. Further, the system as described above preferablycomprises: (d) at least one exogenous target nucleic acid coding for thetarget protein or a variant or mutated form of the target proteinwherein said exogenous target nucleic acid differs from the endogeneoustarget gene on the nucleic acid level such that the expression of theexogenous target nucleic acid is substantially less inhibited by thesilencing agent (e.g. RNAi agent) than the expression of the endogeneoustarget gene.

The test compounds of the present invention can be obtained using any ofthe numerous approaches in combinatorial library methods known in theart, including: biological libraries; spatially addressable parallelsolid phase or solution phase libraries; synthetic library methodsrequiring deconvolution; the ‘one-bead one-compound’ library method; andsynthetic library methods using affinity chromatography selection. Thebiological library approach is limited to peptide libraries, while theother four approaches are applicable to peptide, non-peptide oligomer orsmall molecule libraries of compounds (Lam, K. S. (1997) Anticancer DrugDes. 12:145).

Examples of methods for the synthesis of molecular libraries can befound in the art, for example in: DeWitt et al. (1993) Proc. Natl. Acad.Sci. U.S.A. 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA91:11422; Zuckermann et al. (1994). J. Med. Chem. 37:2678; Cho et al.(1993) Science 261:1303; Carrell et al. (1994) Angew. Chem. Int. Ed.Engl. 33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061;and in Gallop et al. (1994) J. Med. Chem. 37:1233.

Libraries of compounds may be presented in solution (e.g., Houghten(1992) Biotechniques 13:412-421), or on beads (Lam (1991) Nature354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria (LadnerU.S. Pat. No. 5,223,409), spores (Ladner USP '409), plasmids (Cull etal. (1992) Proc Natl Acad Sci USA 89:1865-1869) or on phage (Scott andSmith (1990) Science 249:386-390); (Devlin (1990) Science 249:404-406);(Cwirla et al. (1990) Proc. Natl. Acad. Sci. 87:6378-6382); (Felici(1991) J. Mol. Biol. 222:301-310); (Ladner supra.)).

In a preferred embodiment, the library is a natural product library,e.g., a library produced by a bacterial, fungal, or yeast culture. Inanother preferred embodiment, the library is a synthetic compoundlibrary.

When used in the screening assays described herein, the RNA silencingagents of the invention offer improvements in potency over existing RNAsilencing agents. For example, the RNA silencing agents of the inventionhave several-fold improvement in potency (picomolar vs. micromolar) suchthat lower and more economical amounts of these reagents can be used forthe methods described supra. Alternatively, the use of the RNA silencingagents of the invention may obviate the need for costly chemicalmodifications.

This invention is further illustrated by the following examples whichshould not be construed as limiting. The contents of all references,patents and published patent applications cited throughout thisapplication are incorporated herein by reference.

EXAMPLES Example I Functionally Asymmetric siRNA Duplexes

To assess quantitatively if the two strands of an siRNA duplex areequally competent to direct RNAi, the individual rates of sense andanti-sense target cleavage for an siRNA duplex directed against thefirefly luciferase mRNA were examined (FIG. 1A). The relevant portionsof the sense and anti-sense target RNA sequences are shown in FIG. 1Aand the siRNA sequence in FIG. 1B. This siRNA duplex effectivelysilences firefly luciferase expression in culture human HeLa cells.Using a Drosophila embryo-derived in vitro RNAi reaction, a significantdifference in the rate of target cleavage for the two siRNA strands wasfound; the anti-sense siRNA strand directed more efficient RNAi againsta sense RNA target than the sense siRNA strand for an anti-sense target(FIG. 1B). (Anti-sense siRNA strands and sense target RNAs are alwaysshown in black, and sense siRNAs and anti-sense targets, in grey).Control experiments showed that using siRNA duplexes with 5′ phosphatesdid not alter this result (data not shown), indicating that differentrates of phosphorylation for the two strands is not the cause for theobserved asymmetry. Surprisingly, the two stands of the luciferaseduplex siRNA duplex, used individually as 5′ phosphorylated singlestands, had identical rates of target cleavage (FIG. 1C). RNAi directedby single-stranded siRNA is roughly 10-fold less efficient than thattriggered by siRNA duplexes, reflecting the ˜100-fold lower stability ofsingle-stranded siRNAs in vitro and in vivo (Schwarz et al., 2002). Thedifference in the rate of cleavage directed by the sense and anti-sensestrands when the reaction was programmed with an siRNA duplex isunlikely to reflect a difference in the inherent susceptibility of thetwo targets to RNAi. Instead, the observation that the same two siRNAstrands are equally effective as single-strands, but show dramaticallydifferent activities when paired with each other, indicates that theasymmetry in their function is established at a step in the RNAi pathwayprior to the encounter of the programmed RISC with its corresponding RNAtarget.

Example II Differential RISC Assembly Accounts for siRNA StrandFunctional Asymmetry

To identify the source of asymmetry in the function of this siRNAduplex, the unwinding of the two siRNA strands when the duplex wasincubated in a standard in vitro RNAi reaction was measured. This assaywas shown previously to determine accurately the fraction of siRNA thatis unwound in an ATP-dependent step in the RNAi pathway; no functionalRISC is assembled in the absence of ATP (Nykänen et al., 2001). Previousstudies show that siRNA unwinding correlates with capacity of an siRNAto function in target cleavage (Nykänen et al., 2001; Martinez et al.,2002), demonstrating that siRNA duplex unwinding is required to assemblea RISC competent to base pair with its target RNA. Here, theaccumulation of single stranded siRNA from the luciferase siRNA duplexafter 1 hour incubation in an in vitro RNAi reaction in the absence oftarget RNA was measured. After one hour of incubation with Drosophilaembryo lysate in a standard RNAi reaction, 22% of the anti-sense strandof the luciferase siRNA was converted to single-strand (FIG. 1D; ‘siRNAB’ solid black bar). Remarkably, a corresponding amount ofsingle-stranded sense siRNA was not detected. Instead, only 3% of thesense strand accumulated as single-stranded siRNA (FIG. 1D; ‘siRNA B’solid grey bar). In control experiments, no single-stranded RNA wasdetected without incubation in lysate (not shown), demonstrating thatthe siRNA was entirely double-stranded at the beginning of the reaction.Since the production of single-stranded anti-sense siRNA must beaccompanied by an equal amount of single-stranded sense siRNA, themissing sense-strand must have been destroyed after unwinding.

To establish that the observed asymmetry in the accumulation of the twosingle-strands was not an artifact of our unwinding assay, anindependent method for measuring the fraction of siRNA present assingle-strands in protein-RNA complexes was. In this assay,double-stranded siRNA was incubated with Drosophila embryo lysate in astandard RNAi reaction for 1 h, then a 31 nt 2′-O-methyl RNAoligonucleotide containing a 21 nt sequence complementary to theradiolabeled siRNA strand was added. 2′-O-methyl oligonucleotides arenot cleaved by the RNAi machinery, but can bind stably to complementarysiRNA within the RISC (Martin Simard, GH, Craig Mello, and PDZ,manuscript in preparation). To allow recovery of RISC, the 2′-β-methyloligonucleotide was tethered to a magnetic bead via abiotin-streptavidin linkage. After washing away unbound RNA and protein,the amount of radioactive siRNA bound to the bead was measured. Theassay was performed with separate siRNA duplexes in which either thesense or the anti-sense strand was 5′-³²P-radiolabeled. Capture of³²P-siRNA was observed when the 2′-O-methyl oligonucleotide contained a21-nt region complementary to the radiolabeled siRNA strand, but notwhen an unrelated oligonucleotide was used. The assay captures all RISCactivity directed by the siRNA strand complementary to the tetheredoligonucleotide, demonstrating that it measures siRNA present in thelysate as single-strand complexed with RISC proteins. This assayrecapitulates the results of the unwinding assay described above: forthe siRNA in FIG. 1D; ‘si RNA B’ open bars, nearly ten-fold moreanti-sense siRNA was detected than sense strand. An explanation forthese results is that the two strands of this siRNA duplex aredifferentially loaded into the RISC, and that single-stranded siRNA notassembled into RISC is degraded. Functional asymmetry occurred only whenthe trigger siRNA was double-stranded, not when the two siRNA strandswere tested individually (FIGS. 1B and 1C). Thus, asymmetric assembly ofRISC was a feature of the siRNA duplex, rather than of either thesequences of the individual siRNA strands or the accessibility of thetargeted sites to cleavage.

Example III Base-Pairing at the 5′ End of the siRNA Strand Gates RISCAssembly

The finding that the two siRNA strands can have different capacities toform RISC when paired in a duplex indicates that some feature of the 19base-pairs of the duplex determines functional asymmetry. Thesebase-pairs must be disrupted to produce RISC (Nykänen et al., 2001),which contains single-stranded siRNA (Martinez et al., 2002). The siRNAsused in FIG. 1B were examined for base-pairing features that mightdistinguish the two siRNA strands. For the siRNA in FIG. 1B, the 5′ endof the anti-sense siRNA strand begins with U and is thus paired to thesense siRNA strand by an A:U base pair (2 hydrogen bonds). In contrast,the 5′ nucleotide of the sense siRNA strand is linked to the anti-sensestrand by a C:G base pair (3 hydrogen bonds). The sense siRNA strandforms 8-10-fold less RISC and guides cleavage of its RNA target at acorrespondingly slower rate than the anti-sense strand. A workinghypothesis to explain the observed functional asymmetry is that thesiRNA strand whose 5′ end is more weakly bound to the complementarystrand more readily incorporates into RISC. In this view, the relativebase-pairing strengths of the 5′ ends of the two siRNA strands woulddetermine their relative extents of RISC formation.

As an initial test of this idea, the 5′ nucleotide of the siRNA sensestrand was changed from C to U (FIG. 1E). This changed the base pairformed between the 5′ most nucleotide of the sense strand and position19 of the anti-sense strand from the Watson-Crick base pair C:G to theweaker, less stable wobble pair U:G, while leaving the anti-sense strandof the siRNA unaltered. Remarkably, the change of this single nucleotidenot only enhanced the rate of cleavage directed by the sense strand, butvirtually eliminated the ability of the anti-sense strand to direct RNAi(FIG. 1E).

To determine the basis for the reversed functional asymmetry for thesiRNA in FIG. 1E, the amount of each strand that was single strandedafter incubation of the siRNA duplex in Drosphilia embryo lysate wasdetermined. After 1 h, nearly 30% of the sense siRNA strand wasconverted to single stranded, but no single-stranded anti-sense strandwas detected (FIG. 1D; ‘siRNA E’). Therefore, the simplest explanationfor the asymmetric function of this siRNA is that the sense strand, butnot the anti-sense, of this siRNa duplex was incorporated into RISC.Thus, a single nucleotide mutation in the sense siRNA strand of thesiRNA in FIG. 1B completely reversed the relative abilities of the twostrands to assemble in the enzyme complex that directs RNAi.

The stability of the initial five base pairs of the siRNA strands wascalculated in FIG. 1 using the nearest-neighbor method and the mfoldalgorithm (D. H. Mathews, 1999; Zuker, 2003). The 5′ end of the sensesiRNA strand in FIG. 1E, but not that in 1B, is predicted to exist as anequilibrium of two conformers of nearly equal energy (FIG. 10). In oneconformer, the 5′ nucleotide of the sense strand is bound to theanti-sense strand by a U:G wobble pair, whereas in the other conformerthe 5′ end of this siRNA strand is unpaired. The analysis suggests thatRISC assembly favors the siRNA strand whose 5′ end has a greaterpropensity to fray.

To test this hypothesis further, the strand-specific rates of cleavageof sense and anti-sense human Cu, Zn superoxide dismutase-1 (sod1) RNAtargets (FIG. 3A) triggered by the siRNA duplex shown in FIG. 3B wereexamined. Given that the 5′ ends of both siRNA strands of this duplexare in G:C base pairs, it was anticipated that this duplex would notdisplay pronounced target cleavage asymmetry. As shown in FIG. 3B, thetwo strands are similar in their rates of target cleavage, although therate of anti-sense cleavage directed by the sense-strand is clearlyfaster than the rate of sense-target cleavage guided by the anti-sensestrand. This small difference in rate is likely explained by thesense-strand forming 20 base pairs with its target RNA, whereas theanti-sense strand can form only 19, consistent with previous reportsthat the penultimate position of an siRNA makes a small contribution toits efficacy (Elbashir et al., 2001b). Next, the C at position 19 of thesense strand was changed to A, causing the anti-sense strand to beginwith an unpaired nucleotide. This change, which was made to thesense-strand of the siRNA, caused the rate of target cleavage guided bythe anti-sense siRNA strand to be dramatically enhanced and the sensestrand rate to be suppressed (FIG. 3C). Because the enhancement of sensetarget cleavage was caused by a mutation in the sense siRNA strand,which does not participate in the recognition of the sense target, theeffect of the mutation must be on a step in the RNAi pathway that isspatially or temporally coupled to siRNA unwinding. However, thesuppression of anti-sense target cleavage clearly might have resultedfrom the single-nucleotide mismatch between the sense strand and itstarget RNA generated by the C-to-U substitution.

To test if the suppression of the rate of anti-sense target cleavage wasa consequence of the position 19 mismatch, a different strategy was usedto unpair the 5′ end of the anti-sense strand. FIG. 3D shows an siRNA inwhich the sense-strand is identical to that in FIG. 3B, but the firstnucleotide of the anti-sense strand has been changed from G to U,creating a U-C mismatch at its 5′ end, in place of the G-A of FIG. 3C.Nonetheless, this siRNA duplex showed pronounced asymmetry, with theanti-sense strand guiding target cleavage to the nearly completeexclusion of the sense strand (FIG. 3D). Thus, the suppression of thecleavage rate of the sense-strand in FIG. 3C was not a consequence ofthe position 19 mismatch. This finding is consistent with previousstudies that suggest that mismatches with the target RNA are welltolerated if they occur near the 3′ end of the siRNA guide strand(Amarzguioui et al., 2003). The finding that the siRNAs in FIGS. 3C and3D display profound asymmetry demonstrates that both the enhancement ofthe target cleavage rate of the anti-sense strand and the suppression ofthe function of the sense strand is a consequence of their relativeabilities to enter the RNAi pathway, not their intrinsic capacity todirect target cleavage.

Finally, the sense strand of FIG. 3C was paired with the anti-sensestrand of FIG. 3D to create the siRNA duplex shown in FIG. 3E. The sensestrand of this siRNA, like that in FIG. 3C, contains a mismatch with theanti-sense target at position 19 Like the anti-sense siRNA strand inFIG. 3D, the anti-sense strand contains a mismatch with the sense targetat position 1. This siRNA duplex directs target anti-sense cleavagesignificantly better than the siRNA in FIG. 3C, despite the fact thatthe two siRNAs contain the same sense strand (FIG. 3E).

FIGS. 3F, G, and H show a similar analysis in which the 5′ end of thesense strand or position 19 of the anti-sense strand of the siRNA inFIG. 3B was altered to produce siRNA duplexes in which the 5′ end of thesense strand was either fully unpaired (FIGS. 3F and G) or paired in anA:U base pair (FIG. 3H). Again, unpairing the 5′ end of an siRNAstrand—the sense strand, in this case—caused that strand to function tothe exclusion of the other strand. When the sense strand 5′ end waspresent in an A:U base pair and the anti-sense strand 5′ end was in aG:C pair, the sense strand dominated the reaction (FIG. 3H), althoughnow the anti-sense strand showed activity similar to that seen for theoriginal siRNA (FIG. 3B) in which both strands were in G:C pairs attheir 5′ ends. Converting the unpaired 5′ end of the siRNAs in FIG. 3 toan A:U pair reduced the functional asymmetry of the two strands byenhancing the efficacy of the sense strand (FIG. 3E) or the anti-sensestrand (FIG. 3H). The relative ease with which the 5′ ends of the twosiRNAs can be liberated from the duplex determines the degree ofasymmetry. Additional data supporting this idea is shown in FIG. 8,using a different siRNA. FIG. 8B shows an siRNA that cleaved the twosod1 target RNAs (FIG. 8A), with modest functional asymmetry thatreflects the collective base pairing strength of the first four or fivenucleotides of each siRNA strand (FIG. 8E; see below). Asymmetry wasdramatically increased when a G:U wobble was introduced at the 5′ end ofthe anti-sense strand of the siRNA (FIG. 8C), but no asymmetry was seenwhen the individual single-strands strands were used to trigger RNAi(FIG. 8D), demonstrating that differential RISC assembly, not targetaccessibility, explains the functional asymmetry of the siRNA duplex.

Together, the data in FIGS. 1, 2, and 8 indicates that the symmetry ofRISC assembly is determined by a competition between the fraying of the5′ ends of the two siRNAs in the duplex. Such fraying may initiate adirectional process of unwinding in which the strand at which unwindingis initiated preferentially enters RISC. Such a model requires thateither that RISC assembly factors or RISC components themselves areloaded onto one of the two siRNA strands before unwinding is completed,or that information about the siRNA strands prior state of pairing isretained, perhaps by a protein such as the helicase remaining bound to astrand.

Example IV A Single Hydrogen Bond can Determine which Strand of an siRNADuplex Directs RNAi

To explore this hypothesis further, additional changes were made to thesod1-specific siRNA in FIG. 3. These modifications alter the function ofthe two strands of the siRNA, but do not change the site cleaved on thetwo target RNA's. In FIG. 3A, the anti-sense strand of FIG. 3B waspaired with a sense strand identical to that in FIG. 3B except the 5′ Gwas replace with inosine (I). Like G, I pairs with C, but makes twoinstead of three hydrogen bonds. In this respect, an I:C pair is similarin energy to an A:U pair. The resulting siRNA was functionallyasymmetric, when the sense-strand began with an I, it directed targetcleavage more efficiently than antisense-strand (FIG. 4A). The asymmetryreflects an enhancement in efficacy of the sense siRNA strand, withlittle loss in the function of the anti-sense strand. An inosine at the5′ end of the anti-sense strand had the opposite effect. When the G atposition 1 of the anti-sense strand was substituted with inosine and thesense strand is that of FIG. 3B, the anti-sense strand was enhancedrelative to the sense strand (FIG. 4B). Thus, the strand whose 5′ end isin the weaker base pair was more effective at target cleavage.

Remarkably, when the 5′ nucleotides of both siRNA strands engage in I:Cbase pairs (FIG. 4C), the relative efficacy of the two siRNA strands isrestored to that reported in FIG. 3B. The slightly faster rate foranti-sense target cleavage than for sense target cleavage is also seenfor RNAi triggered with the individual, inosine-containing singlestrands, indicating that it reflects a difference in the intrinsiccapacity of the two strands to guide cleavage, rather than a differencein RISC assembly. Although the relative rates of cleavage of the twostrands are comparable for the siRNAs in FIGS. 3B and 4C, the absoluterates are faster for the siRNA in FIG. 4C. These data indicate thatproduction of RISC from an individual strand is governed both by therelative propensity of the siRNA 5′ end to fray compared to that of itscomplementary strand and by the absolute propensity of the siRNA 5′ endto fray. This latter finding is particularly unexpected, in that itshows that a difference of a single hydrogen bond has a marked effect onthe rate of RISC assembly. siRNA end fraying provides an entry site foran ATP-dependent RNA helicase that unwinds siRNA duplexes (FIG. 4). Thehelicase makes many abortive attempts to dissociate the two siRNAstrands before succeeding to load one strand into RISC. The involvementof a helicase in RISC assembly is supported by previous observations:(1) both siRNA unwinding and production of functional RISC require ATPin vitro (Nykänen et al., 2001) and (2) several proteins with sequencehomology to ATP-dependent RNA helicases have been implicated in RNAsilencing (Wu-Scharf et al., 2000; Dalmay et al., 2001; Hutvàgner andZamore, 2002; Ishizuka et al., 2002; Kennerdell et al., 2002; Tabara etal., 2002; Tijsterman et al., 2002).

The effect of single-nucleotide mismatches in this region of the siRNA,using a series of siRNAs containing a mismatch at the second, third, orfourth position of each siRNA strand was further tested. The siRNAsbearing G:U wobble pairs at the second, third, or both second and thirdpositions (FIG. 11) was also analyzed. The results of this seriesdemonstrate that mismatches, but not G:U wobbles, at positions 2-4 of ansiRNA strand alter the relative loading of the two siRNA strands intoRISC. Mismatches at position five, have very modest effects on therelative loading of the siRNA strands into RISC (data not shown). Incontrast, the effects of internal mismatches at positions 6-15 cannot beexplained by their influencing the symmetry of RISC assembly (data notshown). In sum, these data are consistent with the action of anon-processive helicase that can bind about four nucleotides of RNA.

Example V Implications of siRNA Asymmetry in miRNA Biogenesis

One implication of the findings presented herein is that although siRNAsare predominantly present as duplexes at steady state in vitro (Nykänenet al., 2001) and perhaps in vivo (Hamilton and Baulcombe, 1999; Djikenget al., 2001), both strands of an siRNA are unlikely to be presentequally in RISC. That is, the strength of the base pairs at the 5′ endsof the two siRNA strands can influence their accumulation assingle-strands. When the 5′ end of one strand is unpaired, thisasymmetry can be nearly absolute. This observation suggested thatasymmetric incorporation into RISC, as a consequence of directionalunwinding from a frayed end of an siRNA duplex, might also explain whymiRNAs accumulate as single strands. Animal miRNAs are derived from thedouble-stranded stem of ˜70 nt stem-loop precursor RNAs (Lee et al.,1993; Pasquinelli et al., 2000; Reinhart et al., 2000; Lagos-Quintana etal., 2001; Lau et al., 2001; Lee and Ambros, 2001; Lagos-Quintana etal., 2002). pre-miRNAs stems are only partially double-stranded; thetypical pre-miRNA contains mismatches, internal loops, and G•U basepairs predicted to distort an A-form RNA helix. miRNAs are generatedfrom pre-miRNAs by the double-stranded RNA-specific endonuclease Dicer(Hutvàgner et al., 2001; Grishok et al., 2001; Ketting et al., 2001). Itwas previously proposed by the instant inventors that miRNAs aresingle-stranded because helical discontinuities constrain Dicer to breakonly two, rather than four, phosphodiester bonds, yielding asingle-stranded miRNA, rather than an siRNA-like duplex (Hutvàgner etal., 2001). Such a mechanism has precedent, because E. coli RNase IIIcan be constrained by helical distortions to make only one or two breaksin an RNA chain (Chelladurai et al., 1993).

An alternative hypothesis is that the Dicer cleaves four phosphodiesterbonds in all of its substrates, both long dsRNA and pre-miRNAs, andalways generates a product with the essential siRNA duplex (Hutvàgnerand Zamore, 2002; Reinhart et al., 2002; Lim et al., 2003b). Thismechanism for miRNA production was originally suggested by Bartel andcolleagues. Using a small RNA cloning strategy to identify mature miRNAsin C. elegans, they recovered small RNAs corresponding to the non-miRNAside of the precursor's stem (Lim et al., 2003b). Although these‘miRNA*’ sequences were recovered at about 100 times lower frequencythan the miRNAs themselves, they could always be paired with thecorresponding miRNA to give ‘miRNA duplexes’ with 2 nt overhanging 3′ends (Lim et al., 2003b). Their data suggest that miRNAs are born asduplexes, but accumulate as single-strands because some subsequentprocess stabilizes the miRNA, destabilizes the miRNA*, or both.

The incorporation of miRNA into RISC is this process. Our results withsiRNA suggest that preferential assembly of a miRNA into the RISC wouldbe accompanied by destruction of the miRNA. If the rate asymmetric RISCassembly was faster than the production of the miRNA duplexes, onlysingle-stranded miRNAs would be observed at steady-state (FIG. 4). Theaccumulation of single-strands and not duplexes for miRNAs would simplybe a consequence of Dicer being significantly less efficient in cleavingpre-miRNAs compared to long dsRNA (Hutvàgner et al., 2001). The rate ofasymmetric RISC assembly might be faster than the production of miRNAduplexes, so only single-stranded miRNAs would be observed atsteady-state. Two key predictions of this hypothesis are that (1)purified Dicer should cleave pre-miRNAs into equal amounts of miRNA andmiRNA* products and (2) pre-miRNA structures should be processed byDicer into duplexes with the 5′ end of the miRNA strand frayed or weaklyhydrogen bonded and the 5′ end of the miRNA* strand more securely basepaired.

A. Dicer Cleaves Pre-let-7 Symmetrically

To begin to test the idea that pre-miRNA are cleaved by Dicer togenerate a product with an essential structure of an siRNA a duplex, weincubated the Drosophila pre-miRNA, pre-let-7, with purified,recombinant Dicer and analyzed the products by Northern hybridizationusing probes specific for either the 5′ side of the precursor stem thatencodes mature let-7 or for products derived from the 3′ side of theprecursor stem (let-7* products). As a control, the let-7 precursor RNAwas incubated in Drosophila embryo lysate, which recapitulates bothpre-let-7 maturation and RNAi in vitro. As previously reported,incubation of pre-let-7 RNA in the lysate produced a single bandcorresponding to authentic let-7, but no let-7* products (Hutvàgner etal., 2001; FIGS. 5A and 5B). In contrast, incubation of pre-let-7 withDicer yielded approximately equal amounts of let-7 and let-7* products.At least three distinct RNAs were generated from each side of the stem,rather than the single band corresponding to mature let-7 observed inthe embryo lysate. Thus, the absence of let-7* in vivo and in the embryolysate reaction cannot be explained by the influence of pre-let-7structure on Dicer.

B. Asymmetric RISC Assembly Explains Why miRNAs are Single-Stranded

If Dicer cleaves both sides of the pre-let-7 stem, then some stepdownstream from Dicer action selects mature let-7 from an siRNA-likeduplex in which let-7 is paired with let-7*. A good candidate for such astep would be the asymmetric incorporation of let-7 into RISC,accompanied by the degradation of let-7*. To test this idea, the siRNAthat might be formed if pre-let-7 were cleaved by Dicer into an siRNAduplex-like structure was deduced. The sequence of this ‘pre-let-7siRNA,’ generated by ‘conceptual dicing,’ is shown in FIG. 6A (seebelow). Notably, the 5′ end of let-7 is unpaired in this duplex, whereasthe 5′ end of the let-7* strand is in an A:U base pair. The resultspresented in FIGS. 2, 3, and 4 suggest that this structure should causethe let-7 strand to enter the RISC to the near exclusion of the let-7*strand, which would consequently be degraded.

C. miRNA Versus miRNA* Selection in Drosophila

This analysis was next extended to the other published Drosophila miRNAgenes (Lagos-Quintana et al., 2001). For each precursor structure, thedouble-strand predicted to be produced by Dicer. These conceptuallydiced duplexes are shown in FIG. 6A. For 23 of the 27 duplexes generatedby this analysis (including pre-let-7), the difference in the basepairing of first five nucleotides of the miRNA versus miRNA* strandsaccurately predicted the miRNA, and not the miRNA,* accumulates in vivo.The analysis succeeded irrespective of which side of the pre-miRNA stemencoded the mature miRNA. This analysis, previous observations thatsingle mismatches in the first four nucleotides of an siRNA strand, aninitial G:U wobble pair, but not internal G:U wobbles, directed theasymmetric incorporation of an siRNA strand into RISC (FIGS. 1, 2, 3, 8,9, and 11). However, no difference was discerned in the propensity tofray of the 5′ ends of the miRNA and * strands for miR-4, miR-5, thethree miR-6-2 paralogs, and miR-10. Therefore, it could not be explainedwhy a particular strand would accumulate as the mature miRNA for thesethree miRNA precursors. miR-5 and miR-10, like other Drosophila miRNAs,were identified by the cloning and sequencing of small RNAs from embryos(Lagos-Quintana et al., 2001). Determinants other than end frayingappear to function in the selection of miR-4 and miR-6; these unknowndeterminants may also play a role in the assembly of an siRNA strandinto RISC. However, miR-5 and miR-10 were cloned only once, raising thepossibility that miR-5* or miR-10* is present in embryos, but notrepresented among the library of small RNA's from which the miRNAs werecloned. Similarly, miR-6 is encoded by three paralogous genes, only oneof which we predict to produce detectable amounts of the miR*, so this *strand might have also gone undetected. To test if both the miRNA and *strands might accumulate for some or all of these three genes, Northernhybridization was used to examined the relative abundance of miR-10 andmiR-10* in adult Drosophila males and females, and in syncitialblastoderm embryos. The results detected both miR-10* and mi-R10 in vivo(FIG. 6C). In fact, the results indicated that more miR-10* was detectedthat miR-10 in adult males. This finding strengthens the proposal thatmiRNA genes (i.e., premiRNA's) uniquely specify on which side of thestem the miRNA residues by generating siRNA-like duplexes from whichonly one of the two strands of the duplex is assembled into RISC. Whenthese double-stranded intermediates do not contain structural featuresenforcing asymmetric RISC assembly, both strands accumulate in vivo. Itis possible that pre-miRNAs such as pre-miR-10, which generates roughlyequal amounts of small RNA products from both sides of the precursorstem, simultaneously regulate target RNAs with partial complementary toboth small RNA products.

Example VI Increased Rate of siRNA Efficiency Through the Use of dTdTTails

Art-recognized protocols for designing siRNA duplexes teach theinclusion of dTdT tails (i.e., 2-nucleotide overhangs consisting ofdTs). Two duplexes were created to test whether the addition of 3′overhanging dTdT tails increases the rate of siRNA targeting efficiencyof the Cu, Zn superoxide-dismutase-1 (Sod1) mRNA. The first duplexcontained sense and antisense stands, each including 21 nucleotides with19 complementary bases plus 2-nucleotide overhangs (the overhangsconsisting of bases in common with the target sequence). The secondduplex contained sense and antisense strands, each including 19complementary nucleotides (in common with the Sod1 target), plus2-nucleotide dTdT tails at the 5′ end of the strand (not matching theSod1 target). Results demonstrate that the rate of siRNA efficiencyimproved ˜8 fold—when using the duplex having mismatched dTdT tails(FIG. 12).

Discussion of Examples I-VI: Implications for RNA Silencing

The observations described herein provide rules for siRNA design.Clearly, siRNA structure can profoundly influence the entry of theanti-sense siRNA strand into the RNAi pathway. Thus, the sequence of thesiRNA, rather than that of the target site, may explain at least someprevious reports of ineffective siRNAs duplexes. Such inactive duplexesmay be coaxed back to life by modifying the sense strand of the siRNA toreduce the strength of the base pair at the 5′ end of the anti-sensestrand. An example of this in vitro is shown in FIG. 9, for anineffective siRNA directed against the huntingtin (htt) mRNA (FIG. 9A).Changing the G:C (FIG. 9B) to an A:U pair (FIG. 9C) or a G-A mismatch(FIG. 9D) dramatically improved its target cleavage rate in vitro andits efficacy in vivo (Eftim Milkani, NA, and PDZ, unpublishedobservations). In fact, Khvorova and colleagues have found that a lowbase-pairing stability at the 5′ end of the antisense strand, but notthe sense strand, is a prerequisite for siRNA function in culturedmammalian cells (Anastasia Khvorova, Angela Reynolds, and Sumedha D.Jayasena, manuscript submitted).

siRNAs designed to function asymmetrically may also be uses to enhanceRNAi specificity. Recently, expression profiling studies have shown thatthe sense-strand of an siRNA can direct off-target gene silencing (A. L.Jackson, et al. (2003) Nature Biotechnology, May 18; Smizarov et al.,PNAS, 2003; Chi et al., PNAS, 2003). The data presented herein provide astrategy for eliminating such sequence-specific but undesirable effects:redesigning the siRNA so that only the anti-sense strand enters the RNAipathway.

The observations described herein provide new design rules for theconstruction of short hairpin RNAs (shRNAs), which produce siRNAstranscriptionally in cultured cells or in vivo (Brummelkamp et al.,2002; McManus et al., 2002; Paddison et al., 2002; Paul et al., 2002;Sui et al., 2002; Yu et al., 2002). shRNA strategies typically employ aPol III promoter to drive transcription, so the shRNA must begin withseveral G residues. As a consequence, the 5′ end of the siRNA may besequestered in a G:C base pair, significantly reducing entry of theanti-sense strand into the RNAi pathway. To avoid this problem, theanti-sense strand of the desired siRNA can be placed on the 3′ side ofthe loop, so as to ensure that its 5′ end is in an A:U, rather than theG:C pair typically encoded. Alternatively, the hairpin can be designedto place the 5′ end of the anti-sense siRNA strand in a mismatch or G•Ubase pair, in which case it can be placed on either side of the stem.Moreover, a recent report suggests that some shRNAs may induce theinterferon response (Bridge et al., 2003). The data suggest thatmismatches and G:U pairs could be designed into these shRNAssimultaneously to promote entry of the correct siRNA strand into theRNAi pathway and to diminish the capacity of the shRNA stem to triggernon-sequence specific responses to double-stranded RNA.

Finally, the data identify an unanticipated step in the RNAi pathway:the direct coupling of siRNA unwinding to RISC assembly. This findingsuggests that the helicase responsible for unwinding siRNA duplexes willbe intimately linked to other components of the RNAi machinery.Identifying the helicase and the proteins with which it functions toassemble the RISC is clearly an important challenge for the future.

Example VII The siRNA-Programmed RISC is an Enzyme

RISC programmed with small RNA in vivo catalyzes the destruction oftarget RNA in vitro without consuming its small RNA guide (Tang et al.,2003) (Hutvàgner et al., 2002). To begin a kinetic analysis of RISC, theRISC programmed in vitro with siRNA is likewise a multiple-turnoverenzyme was first confirmed. To engineer an RNAi reaction that containeda high substrate concentration relative to RISC, an siRNA was used inwhich the guide strand is identical to the let-7 miRNA, but unlike themiRNA, the let-7 siRNA is paired to an RNA strand anti-sense to let-7(Hutvàgner et al., 2002). The let-7 strand of this siRNA has a highintrinsic cleaving activity, but a reduced efficiency of incorporationinto RISC (FIG. 19A).

After incubating the let-7 siRNA with Drosophila embryo lysate in thepresence of ATP, RISC assembly was inactivated by treatment with N-ethylmaleimide (NEM), and the amount of RISC generated was measured using thepreviously described tethered 2′-O-methyl oligonucleotide assay(Hutvàgner et al., 2004; Schwartz et al., 2003) (FIG. 19 B,C). Theamount of let-7 programmed RISC increased with increasing siRNAconcentration, until the assembly reaction began to saturate at ±50 nM,reaching an asymptote between 3 and 4 nM RISC. Using 0.6 nM RISC, >50cycles of target recognition and cleavage per enzyme complex (data notshown) was observed, confirming that siRNA-programmed RISC is amultiple-turnover enzyme.

Example VIII Multiple-Turnover is Limited by Product Release

The evaluation of the kinetics of siRNA-directed target cleavage in thepresence or absence of ATP was further performed (FIG. 13). RISC wasassembled in the presence of ATP, then the energy regenerating enzyme,creatine kinase, was inactivated with NEM, and ATP depleted by addinghexokinase and glucose (ATP conditions). For +ATP measurements creatinekinase was added to the reaction after NEM-treatment, and the hexokinasetreatment was omitted. A faster rate of cleavage in the presence than inthe absence of ATP was observed. This difference was only apparent latein the reaction time course, indicating that the ATP-dependent rate ofcleavage was faster than the ATP-independent rate only at steady state(FIG. 13 A). The analysis was repeated in more detail (FIG. 13 B). Inthe absence of ATP, a burst of cleaved product early in the reaction,followed by a ˜4-fold slower rate of target cleavage was observed. Noburst was observed in the presence of ATP (FIG. 13A). If the burstcorresponds to a single-turnover of enzyme, then extrapolation of theslower steady state rate back to the y-axis should give the amount ofactive enzyme in the reaction. The y-intercept at the start of thereaction for the steady-state rate was 4.9 nM, in good agreement withthe amount of RISC estimated using the tethered 2′-O-methyloligonucleotide assay (˜4 nM; FIG. 13 B).

In principle, ATP could enhance target recognition by RISC, promote arearrangement of the RISC/target complex to an active form, facilitatecleavage itself, promote the release of the cleavage products from thesiRNA guide strand, or help restore RISC to a catalytically competentstate after product release. All of these steps, except product releaseand restoration to catalytic competence, should affect the rate of bothmultiple and single-turnover reactions. Therefore, the rate of reactionin the presence and in the absence of ATP under conditions in which RISCwas in excess over the RNA target was analyzed. At early times underthese conditions, the reaction rate should reflect only single-turnovercleavage events, in which events after cleavage do not determine therate of reaction. Using single-turnover reaction conditions, identicalrates of RISC-mediated cleavage in the presence or absence of ATP wasobserved (FIG. 13 C). Thus, ATP must enhance a step that occurs onlywhen each RISC catalyzes multiple cycles of target cleavage.

If product release is rate-determining for multiple-turnover catalysisby RISC in the absence, but not the presence, of ATP, then modificationsthat weaken the strength of pairing to the target RNA might enhanceproduct release, but would not be expected to accelerate the return ofthe RISC to a catalytically competent state. Mismatches between thesiRNA and its RNA target at the 3′ end of the siRNA guide strand wasincorporated and designed the siRNAs to be functionally asymmetric,ensuring efficient and predictable incorporation of the let-7 strandinto RISC (FIG. 14 A). The reaction velocity under conditions ofsubstrate excess in the presence and in the absence of ATP for siRNAswith zero to four mismatches between the guide strand 3′ end and the RNAtarget were compared. Cleavage was measured from 100 and 540 s,when >90% of the target remained uncleaved, ensuring that themultiple-turnover reaction was at steady state. Even a single 3′mismatch between the siRNA and its target increased the ATP rate,relative to the +ATP rate, and siRNAs with two or more mismatches showedno significant difference in rate between the presence and absence ofATP (FIG. 14B). The results indicated that in the absence of ATP,product release is the rate-determining step for siRNAs fully matched totheir RNA targets.

Example IX siRNA:Target Complementarity and RISC Function

Mismatches between the siRNA and its target facilitate product release,but not without cost: the rate of reaction, irrespective of ATPconcentration, decreases with each additional 3′ mismatch. When theconcentration of RISC was ˜16-80-fold greater than the target RNAconcentration, each additional mismatch between the 3′ end of the siRNAguide strand and the RNA target further slowed the reaction (FIG. 14C,D). Under conditions of substrate excess, the effect of mismatchesbetween the 3′ end of the siRNA guide strand and its RNA target was morestriking (FIG. 15A): the rate of cleavage slowed ˜20% for eachadditional mismatch. To test the limits of the tolerance of RISC for 3′mismatches, cleavage under modest (8-fold, FIG. 15 B) and vast(˜80-fold, FIGS. 15 C and 16 C) enzyme excess over target RNA wasanalyzed. Remarkably, cleavage was detected for siRNAs with as many asnine 3′ mismatches to the RNA target (FIGS. 15 C and 16 C), but onlyafter 24 hour incubation. No cleavage was detected for an siRNA with ten3′ mismatches to the RNA target (FIG. 15 C).

Linsley and colleagues have proposed siRNA-directed down-regulation ofan mRNA with as few as eleven contiguous bases complementary to thesiRNA guide strand (Jackson et al., 2003). In that study, the mRNAtarget paired with both nts 2-5 and nts 7-17 of the siRNA guide strand,but mismatched at nts 1 and 6 of the siRNA. Results indicated that up tofive mismatched bases are tolerated between the 5′ end of the siRNA andits RNA target (FIG. 16 A,B). No cleavage was detected for siRNAs withsix, seven, or eight 5′ mismatches to the target, even after 24 hourincubation. The siRNA bearing eight mismatches between its 5′ end andthe let-7 complementary target was fully active when eight compensatorymutations were introduced into the let-7 binding site (FIGS. 15C and16B), demonstrating that mutation of the siRNA was not the cause for itsinactivity against the mismatched target. Similarly, when eightmismatches with the 3′ or 5′ end of the siRNA were created by changingthe sequence of the RNA target, target RNA cleavage when the targetcontained eight mismatches with the siRNA 3′ end, but not with the 5′end was detected (FIG. 16 B,C).

To begin to estimate the minimal number of base pairs between the siRNAand its target that permit detectable cleavage by RISC at 24 hourincubation, seven, eight, or nine 3′ mismatches with increasing numbersof 5′ mismatches were combined (FIG. 16 C). Cleavage was detected for asmany as nine 3′ mismatches. However, no detectable cleavage occurredwhen seven, eight, or nine 3′ mismatches were combined with two or more5′ mismatches. In contrast, a single 5′ mismatch (p1) enhanced targetcleavage directed by all three 3′ mismatched siRNAs. Only 6% of thetarget RNA was cleaved after 24 hours when the siRNA contained ninecontiguous 3′ mismatches with the target RNA, but 10% was cleaved whenthe siRNA contained both nine 3′ mismatches and a single (p1) 5′mismatch. Cleavage was similarly enhanced by the addition of a p1mismatch to seven 3′ mismatches (49% cleavage versus 75% cleavage at 24hours) or to eight 3′ mismatches (21% versus 42% cleavage at 24 hours).The finding that unpairing of the first base of the siRNA guide strandpotentiated cleavage under single-turnover conditions indicated that aconformational change occurs in RISC during which the paired p1 basebecomes unpaired prior to cleavage. Intriguingly, p1 is often predictedto be unpaired for miRNAs bound to their targets (Lewis et al., 2003;Rhoades et al., 2002; Stark et al., 2003).

For siRNAs that pair fully with their RNA targets, the scissilephosphate always lies between the target nucleotides that pair withsiRNA bases 10 and 11 (Elbashir et al., 2001; Elbashir et al., 2001).Analysis at single nucleotide resolution of the 5′ cleavage productsgenerated by siRNAs with three, four, or five 5′ mismatches (FIG. 16 D)or six 3′ terminal mismatches (data not shown) revealed that thescissile phosphate on the target RNA remained the same, even when five5′ nts of the siRNA guide strand were mismatched with the target RNA(FIG. 16 D). As discussed below, this result indicates that the identityof the scissile phosphate is a consequence of the structure of RISC,rather than being measured from the 5′ end of the helix formed betweenthe siRNA and its RNA target.

Example X Kinetic Analysis of RISC Catalysis

The role of nucleotides in the terminal regions of the siRNA guidestrand in directing RISC activity was next studied. Reduced pairingbetween an siRNA and its target might disrupt the binding of RISC to itstarget. Alternatively, mismatches might disrupt the structure, but notthe affinity, of the siRNA/target interaction. Fully matched siRNAs arethought to form a 21 base-pair, A-form helix with the target RNA (Chiuet al., 2003; Shiu et al., 2002), but do all parts of this helixcontribute equally to target binding or do some regions provide only acatalytically permissive geometry? To distinguish between thesepossibilities, the Michaelis-Menten kinetics of siRNA-directedtarget-RNA cleavage for a perfectly matched siRNA and for three siRNAsmismatched at their termini was analyzed. siRNAs were assembled intoRISC, then diluted with reaction buffer to the desired RISCconcentration and mixed with target RNA. For each siRNA, the initialvelocity of reaction was determined at multiple substrate concentrations(FIG. 21A), and KM and kcat determined from a non-linear least squaresfit of substrate concentration versus initial velocity (FIG. 17 A). Bythis assay, the KM of the let-7 siRNA with complete complementarity toits target was ˜8.4 nM was estimated (Table 1). A significant differencein KM, within error, between the fully paired siRNA and siRNA variantsbearing three to five mismatches at their 3′ end or three mismatches attheir 5′ end was not detected (FIG. 17A and Table 1). For the mismatchedsiRNAs a higher than optimal enzyme concentration in order to detectcleavage was used. Therefore, the KM measurements for the mismatchedsiRNAs represent an upper bound for the actual KM values.

While the KM was unaltered for the let-7 siRNA containing severalterminal mismatches, the turnover number, kcat, was decreased byterminal mismatches (Table 1). Three mismatches at either the 3′ or the5′ end of the siRNA halved the kcat. The introduction of five, 3′mismatches also had no significant effect on KM, yet decreased kcatnearly 17-fold (Table 1).

Table 1 Summarizes the kinetic data from the analysis in FIG. 17A. Forcomparison, the KM and kcat values of four well studied protein enzymesare provided. KM and kcat±error of fit are reported.

Example XI KM Reflects the Binding Strength of RISC

To estimate the contribution of binding to KM, a competition assay thatmeasures the ability of 2′-O-methyl oligonucleotides to inhibit targetcleavage by RISC was used (FIG. 17 B,C). Such a strategy was usedpreviously to analyze the mechanism of target destruction by antisenseoligonucleotides that recruit RNase H (Lima et al., 1997). Theanticipation was that 2′-O-methyl oligonucleotides would act ascompetitive inhibitors of RISC, because they bind to RISC containingcomplementary siRNA but not to RISC containing unrelated siRNA(Hutvàgner et al., 2004; Meister et al., 2004). Thirty-one nt long,2′-O-methyl oligonucleotides were designed as described previously(Hutvàgner et al., 2004), taking care to exclude sequences predicted toform stable internal structures. 2′-O-methyl oligonucleotides werechosen because of their marked stability in Drosophila lysate andbecause they can be added to the reaction at high μM concentration.

Competition by 2′-O-methyl oligonucleotides and bona fide RNA targetswas quantitatively similar. The reaction velocities of siRNA-directedcleavage of a 32P-radiolabeled target in the presence of increasingconcentrations of unlabeled capped RNA target or a 31-nt 2′-O-methyloligonucleotide corresponding to the region of the target containing thesiRNA binding site was analyzed (FIG. 17 B). Lineweaver-Burk analysis ofthe data confirm that 2′-O-methyl oligonucleotides act as competitiveinhibitors of RISC (data not shown). These data were used to calculateKi values for the perfectly matched RNA and 2′-O-methyl competitors. Forthe capped RNA competitor, the Ki was ˜7.7±4 nM (FIG. 17 B), nearlyidentical to the KM for this siRNA, 8.4 nM (Table 1). The Ki for theperfectly matched 2′-O-methyl competitor oligonucleotide was 3.2±1 nM(FIG. 17 B), essentially the same, within error, as that of the all-RNAcompetitor. The results indicated that 2′-O-methyl oligonucleotides aregood models for 5′-capped RNA targets and that the KM for targetcleavage by RISC is largely determined by the affinity (KD) of RISC forits target RNA.

Although targets with more than five contiguous mismatches to either endof the siRNA are poor substrates for cleavage, they might nonethelessbind RISC and compete with the 32P-radiolabeled target RNA. The2′-O-methyl oligonucleotide competition assay to determine the Ki valuesfor oligonucleotides containing as many as eight mismatches to the siRNAguide strand was used (FIG. 17 B). 2′-O-methyl oligonucleotides with 3′terminal mismatches to the siRNA were good competitors: a fournucleotide mismatch with the 3′ end of the siRNA increased the Ki byonly ˜3-fold (9.0±0.9 nM) and an eight nucleotide mismatch with the 3′end of the siRNA increased the Ki by ˜10-fold (34.8±7 nM). In contrast,mismatches with the 5′ end of the siRNA had a dramatic effect onbinding. A four nucleotide mismatch to the 5′ end of the siRNA increasedthe Ki ˜12-fold (36.4±9.2 nM) and an eight nucleotide mismatch to the 5′end of the siRNA increased the Ki 53-fold (173±16 nM). The differentialeffect on binding between 5′ and 3′ mismatches was maintained even atthe center of the siRNA: a 2′-O-methyl oligonucleotide bearing fourmismatches with siRNA nucleotides 11, 12, 13, and 14 (4 nt 3′ centralmismatch, FIG. 17 B) bound more tightly to RISC (i.e., had a lower Ki)than an oligonucleotide with four mismatches to siRNA positions 7, 8, 9,and 10 (4 nt 5′ central mismatch, FIG. 17 B).

Discussion of Examples VII-XI:

RISC programmed with exogenous siRNA is an enzyme, capable of multiplerounds of target cleavage. Previous studies showed that cleavage of atarget RNA by RISC does not require ATP (Nykänen et al., 2001; Tomari etal., 2004). The more detailed kinetic analysis presented hereinindicates that there are no ATP-assisted steps in either targetrecognition or cleavage by Drosophila RISC; no difference in rate in thepresence or absence of ATP for RNAi reactions analyzed under conditionsof substrate excess at early time points (pre-steady state) or underconditions of enzyme excess where the reaction was essentiallysingle-turnover was detected. In contrast, the steady-state rate ofcleavage under multiple turnover conditions was enhanced four-fold byATP. The results indicates that release of the products of the RISCendonuclease is rate determining under these conditions in the absenceof ATP, but not in the presence of ATP. The most straightforwardexplanation for this finding is that an ATP-dependent RNA helicasefacilitates the dissociation of the products of target cleavage from theRISC-bound siRNA. The involvement of such an ATP-dependent helicase inRNAi in vivo may explain why siRNAs can be active within a broad rangeof GC content (Reynolds et al., 2004).

In the presence of ATP, siRNA-programmed Drosophila RISC is a classicalMichaelis-Menten enzyme. The guide strand of the siRNA studied here hasthe sequence of let-7, an endogenous miRNA. In vivo, let-7 is notthought to direct mRNA cleavage, but rather is believed to repressproductive translation of its mRNA targets. Nonetheless, the let-7 siRNAis among the most potent of the siRNAs we have studied in vitro andprovides a good model for effective siRNA in general. With a kcat of˜7×10-3 s-1, the let-7 siRNA-programmed RISC was slow compared toenzymes with small molecule substrates (Table 1). The KM for this RISCwas ˜8 nM. Enzymes typically have KM values between 1- and 100-foldgreater than the physiological concentrations of their substrates(Stryer et al., 1981). The results indicate that RISC is no exception:individual abundant mRNA species are present in eukaryotic cells at highpM or low nM concentration. The KM of RISC is likely determinedprimarily by the strength of its interaction with the target RNA,because the KM is nearly identical to the Ki of a non-cleavable2′-O-methyl oligonucleotide inhibitor.

Recently, a study of the kinetic parameters of target RNA cleavage byhuman RISC was described (Martinez et al., 2004). In that study, theminimal active human RISC was highly purified; in this study, DrosophilaRISC activity was measured for the unpurified, intact holo-RISC,believed to be an 80S multi-protein complex (Pham et al., 2004).Different siRNAs were used in the two studies. Nonetheless, the KM andkcat values reported here and for the minimal human RISC are remarkablysimilar: the KM was 2.7-8.4 nM and the kcat was 7.1×10-3 sec-1 for thelet-7 siRNA-programmed Drosophila holo-RISC versus a KM of 1.1-2.3 nMand a kcat of 1.7×10-2 sec-1 for a different siRNA in minimal humanRISC. As in this study, a pre-steady-state burst was observed in theabsence of ATP, consistent with the idea that product release isATP-assisted in vivo.

The ratio of kcat to KM is a classical measure of enzyme efficiency andcorresponds to the second order rate constant for the reaction when theconcentration of substrate is much less than the KM. For the let-7programmed RISC, kcat KM-1 equals ˜8.4×105 M-1-1 (˜8.4×10-4 nM-1 s-1), avalue far slower than the expected rate of collision of RISC-1 withmRNA, =107 M-1 s. It is possible that the rate of catalysis by RISC isconstrained by the rate of conformational changes required for formationof the enzyme-substrate complex or by subsequent conformationalrearrangements required for catalysis. It is possible that siRNAs can bedesigned that significantly improve either the kcat or KM of RISCwithout compromising specificity.

Although siRNAs are typically envisioned to bind their target RNAsthrough 19 to 21 complementary base pairs, we find that the 5′, central,and 3′ regions of the siRNA make distinct contributions to binding andcatalysis (FIG. 18). Measurements of KM and Ki suggest that the 5′nucleotides of the siRNA contribute more to target binding than do the3′ nucleotides. At least for the siRNA examined here, the first threeand the last five nucleotides of a 21 nt siRNA contribute little tobinding. If the KD of RISC bound to its target RNA is essentially itsKM, ˜8 nM, then the free energy (.G°=−RT lin KD) of the let-7-programmedRISC:target interaction is approximately −11 kcal mol 1, considerablyless than the −35 kcal mol-1 (KD ˜10-29) predicted32 for the let-7 RNAbound to a fully complementary RNA in 100 mM K+ and 1.2 mM Mg2+ at 25°C. It is possible that RISC discards potential binding energy by bindingless tightly to its target, an siRNA in RISC gains the ability todiscriminate between well matched and poorly match targets, but only forbases in the 5′ region of the siRNA guide strand.

Mismatches between the central and 3′ regions of an siRNA and its targetRNA reduce kcat far more than mismatches at the 5′ end of the siRNA.These results fit well with recent findings by Doench and Sharp thattranslational repression by siRNA, designed to act like animal miRNA, isdramatically disrupted by mismatches with the 5′ end of the siRNA, butnot with similar mismatches at the 3′ end18. These authors propose thatmiRNA binding is mediated primarily by nucleotides at the 5′ end of thesmall RNA. In fact, complementarity between the 5′ end of miRNAs andtheir targets has been required by all computational approaches forpredicting animal miRNA targets (Rajewsky et al., 2004; Lewis et al.,2003; Stark et al., 2003; Enright et al., 2003). The instant discoverythat central and 3′ siRNA sequences must pair with the target sequencefor effective target cleavage but not for target binding reinforces thisview; both central and 3′ miRNA sequences are usually mismatched withtheir binding sites in their natural targets (Lee et al., 1993; Reinhartet al., 2000; Brennecke et al., 2003; Abrahante et al., 2003; Vella etal., 2004; Xu et al., 2003; Johnston et al., 2003).

Formation of a contiguous A-form helix surrounding the scissilephosphate of the target mRNA has been proposed to be a quality controlstep for RISC-mediated target cleavage (Chiu et al., 2003). The instantinvention discovers that RISC can direct cleavage when the siRNA ispaired with the target RNA only at nts 2-12 of the guide strand,corresponding to one complete turn of an RNA:RNA helix. This region ofthe siRNA includes nts 2-8, which appear to be critical for miRNArecognition of mRNAs targeted for translational repression, plus two ntsflanking either side of the scissile phosphate. The instant inventionfurther discovers unpairing the first nt of the guide strand enhancesthe activity of siRNAs with seven, eight or nine 3′ mismatches to theRNA target is striking, since many miRNAs do not pair with their targetsat this position. Furthermore, such pairing resembles that reported byLinsley and colleagues for siRNA-directed off-target effects in culturedmammalian cells (Jackson et al., 2003).

The requirement for a full turn of a helix may reflect a mechanism of‘quality control’ by RISC. Since RISC can apparently assemble on anysiRNA sequence, it must use the structure of the siRNA paired to itstarget to determine whether or not to cleave. Despite the apparentsurveillance of the structure of the siRNA/target pair, the identity ofthe scissile phosphate is unaltered by extensive mismatch between the 5′end of the siRNA and its target. Yet the scissile phosphate isdetermined by its distance from the 5′ end of the siRNA guide strand(Elbashir et al., 2001; Elbashir et al., 2001). The simplest explanationfor the instant discovery is that the scissile phosphate is identifiedby a protein loaded onto the siRNA during RISC assembly, i.e., beforethe encounter of the RISC with its target RNA.

The remarkable tolerance of RISC for mismatches between the siRNA andits targets—up to nine contiguous 3′ nucleotides—implies that a largenumber off-target genes should be expected for many siRNA sequences whenRISC is present in excess over its RNA targets. However, RISC withextensive mismatches between the siRNA and target are quite slow tocleave, so off-target effects may be minimized by keeping the amount ofRISC as low as possible. These understandings of the molecular basis ofsiRNA-directed gene silencing assist the skilled artisan in creatingsiRNAs designed to balance the competing demands of siRNA efficacy andspecificity.

Experimental Procedures A. General Methods

Drosophila embryo lysate preparation, in vitro RNAi reactions, andcap-labeling of target RNAs using Guanylyl transferase were carried outas previously described (Tuschl et al., 1999; Zamore et al., 2000).Target RNAs were used at ˜5 nM concentration to ensure that reactionsoccurred under single-turnover conditions. Target cleavage under theseconditions was proportionate to siRNA concentrations. Cleavage productsof RNAi reactions were analyzed by electrophoresis on 5% or 8%denaturing acrylamide gels. 5′ end labeling and determination of siRNAunwinding status were according to Nykänen et al. (Nykänen et al., 2001)except that unlabeled competitor RNA was used at 100-fold molar excess.Gels were dried, then exposed to image plates (Fuji), which were scannedwith a Fuji FLA-5000 phosphorimager. Images were analyzed using ImageReader FLA-5000 version 1.0 (Fuji) and Image Gauge version 3.45 or 4.1(Fuji). Data analysis was performed using Excel (Microsoft) and Igor Pro5.0 (Wavemetrics).

B. Drosophila embryo lysate, siRNA labeling with polynucleotide kinase(New England Biolabs), target RNA preparation and labeling with guanylyltransferase were carried out as described (Hutvàgner et al., 2002, Haleyet al., 2003) and the forward primer sequence for 379 nt target mRNA was5′-CGC TAA TAC GAC TCA CTA TAG CAG TTG GCG CCG CGA ACG A-3′ (SEQ ID NO:144), and 5′-GCG TAA TAC GAC TCA CTA TAG TCA CAT CTC ATC TAC CTC C-3(SEQ ID NO: 145) for the 182 nt target. Reverse primers used to generatefully matched and mismatched target RNAs were: 5′-CCC ATT TAG GTG ACACTA TAG ATT TAC ATC GCG TTG AGT GTA GAA CGG TTG TAT AAA AGG TTG AGG TAGTAG GTT GTA TAG TGA AGA GAG GAG TTC ATG ATC AGT G-3′ (SEQ ID NO: 146)(perfect match to let-7); 5′-CCC ATT TAG GTG ACA CTA TAG ATT TAC ATC GCGTTG AGT GTA GAA CGG TTG TAT AAA AGG TTG AGG TAG TAG GTT CAT GCA GGA AGAGAG GAG TTC ATG ATC AGT G-3′ (SEQ ID NO: 147) (7 nt 3′ mismatch); 5′-CCCATT TAG GTG ACA CTA TAG ATT TAC ATC GCG TTG AGT GTA GAA CGG TTG TAT AAAAGG TTG AGG TAG TAG GTA CAU GCA GGA AGA GAG GAG TTC ATG ATC AGT G-3′(SEQ ID NO: 148) (8 nt 3′ mismatch); 5′-CCC ATT TAG GTG ACA CTA TAG ATTTAC ATC GCG TTG AGT GTA GAA CGG TTG TAT AAA AGG TTG AGG TAG TAG GAA CATGCA GGA AGA GAG GAG TTC ATG ATC AGT G-3′ (SEQ ID NO: 149) (9 nt 3′mismatch); 5′-CCC ATT TAG GTG ACA CTA TAG ATT TAC ATC GCG TTG AGT GTAGAA CGG TTG TAT AAA AGG TAC TCC ATC TAG GTT GTA TAG TGA AGA GAG GAG TTCATG ATC AGT G-3′ (SEQ ID NO: 150) (8 nt 5′ mismatch); 5′-CCC ATT TAG GTGACA CTA TAG ATT TAC ATC GCG TTG AGT GTA GAA CGG TTG TAT AAA AGG TAC TCGTAG TAG GTT GTA TAG TGA AGA GAG GAG TTC ATG ATC AGT G-3′ (SEQ ID NO:151) (4 nt 5′ mismatch). In FIGS. 13, 14, 15, 17A, 19 and 20A, thetarget sequence was 613 nt long; 379 nt in FIGS. 16A-C,17B and 20B; and182 nt in FIG. 16D. All siRNAs were deprotected according to themanufacturer's protocol (Dharmacon), 5′-radiolabeled where appropriate,then gel purified on a 15% denaturing polyacrylamide gel. 2′-O-methyloligonucleotides were from Dharmacon. siRNA strands were annealed athigh concentrations and serially diluted into lysis buffer (30 nM HEPESpH 7.4, 100 mM KOAc, and 2 mM MgCl2). Gels were dried and imaged asdescribed (Schwartz et al., 2003). Images were analyzed using ImageGauge 4.1 (Fuji). Initial rates were determined by linear regressionusing Excel X (Microsoft) or Igor Pro 5.01 (Wavemetrics). Kaleidagraph3.6.2 (Synergy Software) was used to determine KM and Ki by globalfitting to the equations: V=(Vmax×S)(KM+S)−1 andV=(Vmax×Ki(app))(Ki(app)+I)−1, where V is velocity, S is target RNAconcentration, and I is the concentration of 2′-O-methyl oligonucleotidecompetitor. Ki was calculated by correcting Ki(app) by the KM andsubstrate concentration, Ki=Ki(app)(1+(S KM−1))−1.

C. siRNA Preparation

Synthetic RNAs (Dharmacon) were deprotected according to themanufacturer's protocol. siRNA strands were annealed (Elbashir et al.,2001a) and used at 50 nM final concentration unless otherwise noted.siRNA single strands were phosphorylated with polynucleotide kinase (NewEngland Biolabs) and 1 mM ATP according to the manufacturer's directionsand used at 500 nM final concentration.

D. Target RNA Preparation

Target RNAs were transcribed with recombinant, histidine-tagged, T7 RNAPolymerase from PCR products as described (Nykänen et al., 2001;Hutvàgner and Zamore, 2002), except for sense sod1 mRNA, which wastranscribed from a plasmid template (Crow et al., 1997) linearized withBam HI. PCR templates for htt sense and anti-sense and sod1 anti-sensetarget RNAs were generated by amplifying 0.1 ng/ml (final concentration)plasmid template encoding htt or sod1 cDNA using the following primerpairs: htt sense target, 5′-GCG TAA TAC GAC TCA CTA TAG GAA CAG TAT GTCTCA GAC ATC-3′ (SEQ ID NO: 152) and 5′-UUCG AAG UAU UCC GCG UAC GU-3′(SEQ ID NO: 153); htt anti-sense target, 5′-GCG TAA TAC GAC TCA CTA TAGGAC AAG CCT AAT TAG TGATGC-3′ (SEQ ID NO: 154) and 5′-GAA CAG TAT GTCTCA GAC ATC-3′ (SEQ ID NO: 155); sod1 anti-sense target, 5′-GCG TAA TACGAC TCA CTA TAG GGC TTT GTT AGC AGC CGG AT-3′ (SEQ ID NO: 156) and5′-GGG AGA CCA CAA CGG TTT CCC-3′ (SEQ ID NO: 157).

Immobilized 2′-O-methyl Oligonucleotide Capture of RISC

The 5′ end of the siRNA strand to be measured was 32 P-radiolabeled withPNK. 10 pmol biotinylated 2′-O-Methyl RNA was immobilized on DynabeadsM280 (Dynal) by incubation in 10 ml lysis buffer containing 2 mM DTT for1 h on ice with the equivalent of 50 ml of the suspension of beadsprovided by the manufacturer. The beads were then washed to removeunbound oligonucleotide. 50 nM siRNA was pre-incubated in a standard 50ml in vitro RNAi reaction for 15 min at 25° C. Then, all of theimmobilized 2′-O-Methyl oligonucleotide was added to the reaction andthe incubation continued for 1 h at 25° C. After incubation, the beadswere rapidly washed three times with lysis buffer containing 0.1% (w/v)NP-40 and 2 mM DTT followed by a wash with the same buffer withoutNP-40. Input and bound radioactivity were determined by scintillationcounting (Beckman). The 5′-biotin moiety was linked via a six-carbonspacer arm. 2′-O-methyl oligonucleotides (IDT) were: 5′-biotin-ACA UUUCGA AGU AUU CCG CGU ACG UGA UGU U-3′ (SEQ ID NO: 158) (to capture thesiRNA sense strand) 5′-biotin-CAU CAC GUA CGC GGA AUA CUU CGA AAU GUCC-3′ (SEQ ID NO: 159) (to capture the anti-sense strand).

mfold Analysis

To model the end of an siRNA, the following 16 nt RNA sequence weresubmitted to mfold 3.1: (37° C., 1 M NaCl): CGU ACU UUU GUA CGU G (SEQID NO: 160), UGU ACU UUU GUA CGU G (SEQ ID NO: 161), and UCG AAU UU UUCGAA A (SEQ ID NO: 162).

Pre-let-7 Processing

Pre-let-7 RNA was incubated with N-terminal histadine-tagged, humanDicer according to the manufacterer's directions (Gene therapy Systems)or in a standard Drosophila embryo in vitro RNAi reaction as describedpreviously (Hutvàgner et al., 2001; Hutvàgner and Zamore, 2002).

Northern Hybridization

Northern hybridization was essentially as described (Hutvàgner et al.,2001). 50 mg total RNA was loaded per lane. 5′ 32 P-radiolabeledsynthetic RNA probes (Dharmacon) were: 5′-ACA AAU UCG GAU CUA CAG GGU-3′(SEQ ID NO: 163) (to detect miR-10) and 5′-AAA CCU CUC UAG AAC CGA AUUU-3′ (SEQ ID NO: 164) (to detect miR-10*). The amount of miR-10 ormiR-10* detected was normalized to the non-specific hybridization of theprobe to 5S rRNA. Normalizing to hybridization of the probe to a knownamount of a miR-10 or miR-10* synthetic RNA control yielded essentiallythe same result.

ATP-Depletion and N-Ethyl Maleimide (NEM) Inhibition

RNAi reactions using Drosophila embryo lysate were as described (Haleyet al., 2003). To compare ‘minus’ and ‘plus’ ATP conditions, sampleswere treated with 10 mM NEM (Pierce) for 10 min at 4° C., then the NEMwas quenched with 11 mM dithiothreitol (DTT). For ATP depletion (−ATP),1 unit of hexokinase and 20 mM (final concentration) glucose were addedand the incubation continued for 30 min at 25° C. For ‘plus’ ATPreactions, 0.05 mg ml-1 (final concentration) creatine kinase andone-tenth volume H2O substituted for hexokinase and glucose. Theaddition of fresh creatine kinase after NEM treatment did not rescue thedefect in RISC assembly, but did restore ATP to high levels (Nykänen etal., 2001). ATP levels were measured using an ATP assay kit (Sigma) anda PhL luminometer (Mediators Diagnostika).

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EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

TABLE 1 Table 1 Kinetic analysis of RISC Mismatches, position K_(M) (nM)V_(max) (nM s⁻¹) [RISC] (nM) k_(cat) (s⁻¹) k_(cat) K_(M) ⁻¹ (nM⁻¹ s⁻¹)fold change, k_(cat) K_(M) ⁻¹ none 8.4 ± 1.6 0.0071 1 7.1 × 10⁻³ 8.4 ×10⁻⁴ 1.00 three nt 3′ 2.7 ± 0.6 0.0022 2 1.1 × 10⁻³ 3.8 × 10⁻⁴ 0.46 fivent 3′ 6.0 ± 1.8 0.0054 2 2.7 × 10⁻⁴ 4.5 × 10⁻⁵ 0.05 three nt 5′ 4.7 ±1.3 0.0063 2 3.2 × 10⁻³ 6.7 × 10⁻⁴ 0.80 Reference enzymes K_(M) (nM)k_(cat) (s⁻¹) k_(cat) K_(M) ⁻¹ (nM⁻¹ s⁻¹) k_(cat) K_(M) ⁻¹ relative toRISC Urease (ref 43) 2.5 × 10⁷ 1 × 10⁴ 4.0 × 10⁻⁴ 0.47 Fumarase (ref 43)5.0 × 10³ 8 × 10² 1.6 × 10⁻¹ 190 Catalase (ref 43) 2.5 × 10⁷ 1 × 10⁷ 4.0× 10⁻¹ 8940 RNase H1 (ref 44) 3.8 × 10¹ 5 × 10² 1.3 × 10⁻³ 0.03 Summaryof kinetic data from the analysis in FIG. 17A. For comparison, the K_(M)and k_(cat) values of four well studied protein enzymes areprovided^(43,44). K_(M) and k_(cat) ± error of fit are reported.

1.-48. (canceled)
 49. A small hairpin RNA (shRNA) comprising nucleotidesequence identical to the sense and antisense strand of a substitutedsiRNA duplex comprising one or more substituted base pairs, the duplexcomprising a sense strand and an antisense strand, each strand having a5′ end and a 3′ end, wherein, and wherein the one or more substitutedbase pairs are within about 5 base pairs from the antisense strand 5′end (AS 5′) and the sense strand 3′ end (S 3′) and are selected from thegroup consisting of a mismatched base pair, a wobble base pair, a basepair comprising a rare nucleotide and a base pair comprising a modifiednucleotide, such that entry of the antisense strand into a RISC complexis promoted relative to the sense strand; such that the antisense strandpreferentially guides cleavage of a target mRNA by the RISC complex. 50.The shRNA of claim 49, wherein the nucleotide sequence identical to thesense strand is upstream of the nucleotide sequence identical to theantisense strand.
 51. The shRNA of claim 49, wherein the nucleotidesequence identical to the antisense strand is upstream of the nucleotidesequence identical to the sense strand.
 52. A vector encoding the shRNAof claim
 49. 53. A cell comprising the vector of claim
 52. 54. The cellof claim 53, which is a mammalian cell.
 55. The cell of claim 53, whichis a human cell.
 56. A transgene encoding the shRNA of claim
 49. 57. TheshRNA of claim 49, wherein the one or more substituted base pairscomprise at least one mismatched base pair.
 58. The shRNA of claim 57,wherein the mismatched base pair is selected from the group consistingof G:A, C:A, C:U, G:G, A:A, C:C and U:U.
 59. The shRNA of claim 57,wherein the mismatched base pair is selected from the group consistingof G:A, C:A, C:T, G:G, A:A, C:C and U:T.
 60. The shRNA of claim 49,wherein the one or more substituted base pairs at least one wobble basepair.
 61. The shRNA of claim 60, wherein the wobble base pair is G:U.62. The shRNA of claim 60, wherein the wobble base pair is G:T.
 63. TheshRNA of claim 49, wherein the one or more substituted base pairscomprise at least one base pair comprising a rare nucleotide.
 64. TheshRNA of claim 63, wherein the rare nucleotide is inosine (I).
 65. TheshRNA of claim 64, wherein the base pair is selected from the groupconsisting of an I:A, I:U and I:C.
 66. The shRNA of claim 49, whereinthe one or more substituted base pairs comprise at least one base paircomprising a modified nucleotide.
 67. The shRNA of claim 66, wherein themodified nucleotide is selected from the group consisting of 2-amino-G,2-amino-A, 2,6-diamino-G, and 2,6-diamino-A.
 68. A pharmaceuticalcomposition comprising an shRNA comprising a substituted siRNA duplexcomprising one or more substituted base pairs and apharmaceutically-acceptable carrier, the duplex comprising a sensestrand and an antisense strand, each strand having a 5′ end and a 3′end, wherein, and wherein the one or more substituted base pairs arewithin about 5 base pairs from the antisense strand 5′ end (AS 5′) andthe sense strand 3′ end (S 3′) and are selected from the groupconsisting of a mismatched base pair, a wobble base pair, a base paircomprising a rare nucleotide and a base pair comprising a modifiednucleotide, such that entry of the antisense strand into a RISC complexis promoted relative to the sense strand; such that the antisense strandpreferentially guides cleavage of a target mRNA by the RISC complex,wherein the target mRNA is expressed in a human cell.